TWI378237B - Apparatus for identifying a single biomolecule, apparatus for identifying biomolecules and method for identifying a single biomolecule - Google Patents

Description

六、發明說明： 【發明所屬之技術領域】 本發明係關於一包括複數個光學偵測裝置之生物分析 系統及用於偵測與分析生物分子之生物分析系統的使用， 而生物分子，例如核酸。在一實施例中’本發明係關於一 生物分析系統，其包括至少一萬個之光學偵測裝置用以監 測，在一些實施例中，大量的螢光團（fluorophore)分子用以 平行地偵測與分析生物分子。 【先前技術】 人類基因體計晝（The Human Genome Project, HGP)刺 激定序處理量的大量增加且導致定序成本相對降低。與每 個基因體13年及近三十億美元之成本相比，定序成本已顯 著降低一確切地，兩個獨立之基因體最近已被完成 (McGuire et al·，317:1687 (2007))。個人基因體代表 在對於病患與健康照護提供者兩者之醫學治療中的典範轉 移(paradigm shift)。藉由管理疾病之基因風險因子，健康照 護提供者可更立即實施預防性藥物與提供定做治療。由於 具有經完成之基因體大銀行，藥物設計與投藥可為更加有 效，將藥物基因學(pharmacogenetics)的初期領域往前推進。 為了使對於個人之定做藥物普及化，美國國家衛生研 究院(NIH)國家人類基因研究所（NhgRI)確定了每個基因 體定序成本自千萬降低到近一千美元之基準。然而，一般 之两通量（high-throughput)毛細電泳與自動化基因體定序 技術不能滿足對於個人基因體定序之增加的需求。此外， 4 于之疋序方法需要複雜與錯誤傾向（error-prone)之影像 擷2與分析步驟。例如，為了獲得多重影像，許多現行技 ^舄要不是陣列就是偵測系統來移動。之後可將所產生之 像依序排列、對齊與分析。影像擷取、處理與分析步驟 白為傾向於錯誤、花費額外時間且需要昂貴的設備。相反 地’不包括移動光學之現行系統通常被限制，由於不大多 數目之谓測單元。最後’現行元件不將要被偵測之分子置 於接近鄰近於一對應之偵測單元，其實質上限制了偵測訊 號的強度。 因此’對於減少核酸定序成本之裝置的需要存在。為 了達到“$ 1000基因體，，範例，元件需能平行定序多個分 子、具有經簡化之設計與製造製程及避免需要現行元件與 方法之複雜與錯誤傾向的掃瞄與影像分析處理。另外，元 件需要具有定序單一分子的能力以避免於群集定序 (clustered sequencing)方法之擴大（例如介於理想複製模板 之序列間的飄移）與定序（在定序模板間之逐步定序反應 (stepwise sequencing reaction)的去相（dephasing))兩者步驟 中之已知不同時期的困難。 【發明内容】 本發明實施例提供一包括複數個光學偵測裝置之生物 分析系統及使用用於核酸偵測之生物分析系統的方法，例 如定序。實施例所提供之生物分析系統具有大規模平行定 序反應（large-scale parallel sequencing reaction)之能力， 1378237 即，同時將大量不同的核酸模板進行定序。各定序反應使 - 用一單一分子為模板（即’單一分子定序）。所提供之元 ， 件也具有經簡化之設計-排除了需要目前元件之複雜、昂貴 = 與錯誤傾向的掃瞄與偵測步驟。本發明所提供之經簡化之 設計與系統功能，係部分基於要被/[貞測之核酸所附著（不 是直接就是例如，藉由一聚合酶分子）之連結位(linker site) 的直接聯繫(direct correspondence)與一或多個偵測單元（例 .. 如，光偵測器），及部分基於介於連結位與偵測單元間之 · 短距離。在一些實施例中，藉由一偵測之大的立體角（s〇Hd φ angle)顯示了介於核酸與偵測單元間的此短距離。 在一方面，提供了在一偵測單元鑑定一單一生物分子 的生物分析系統。生物分析系統可包括複數個光學偵測裝 置’各裝置可包括一具一光偵測器之基板，基板被設置來 偵測散發自生物分子的光。在一些實施例中，光偵測器可 包括電荷耗合元件（charged-coupled device)、互補金屬氧化 半導體（complementary metal-oxide semiconductor，CMOS) 感测器或光二極體(photodiode)其中之一。光學偵測裝置可 暴 更包括一遮蔽薄板(blind sheet)於基板上，遮蔽薄板包括具 -有一直挺的一針孔，及一濾層被提供於遮蔽薄板與基板之 間且被提供於針孔之下，濾層為非平面且被設置來過濾散 發自生物分子的光。在一些實施例中，非平面濾層之部分 可為弧線形、球形、橢圓形或弓形。光學偵測裝Ϊ巧*更包 括—連結位’其被提供接近於針孔，連結位被處理以將該 生物分子置於接近針孔。 在另一方面，提供了在一偵測單元鑑定一單〆生物分[s ] 6 1378237 : 子的生物分析系統。生物分析系統可包括複數個光學偵測 . 裝置，各裝置可包括一具一光偵測器之基板，美板被設置 來偵測散發自生物分子的光。光學偵測裝置可=包括一光 散發層形成於基板上’其中光散發層包括一凹處，及包括 一濾層’其被提供於凹處與基板之間且於一針孔之下，滅 層為非平面且被設置來過濾散發自生物分子的光。在一些 .實施例中，非平面濾層之部分可為弧線形、球形、橢圓形 _ .或弓形。光學偵測裝置也可包括一連結位（nnker site)，其 Φ 被提供接近於凹處與針孔，連結位被處理以將生物分子置 於接近針孔。光學偵測裝置可更包括與基板結合之:激發 光源以提供一用以激發一附著於生物分子之螢光團 (fluorophore)的光源。 在另一方面，另一實施例提供一藉由連結至少—核酸 至一實施例所提之光學偵測裝置的一連結位（不是直接 地，就是藉由與結合至連結位之核酸聚合酶結合）並在一 對應之光偵測器上偵測核酸之偵測核酸的方法。在一些實 _ 施例中，偵測核酸藉由與，例如一經標誌(labeled)的探針雜 • 合。在一些實施例中，藉由於光學偵測裝置上執行核酸定 序來偵測核酸。在一些實施例中，核酸定序方法係擇自驗 基延伸定序（base-extension sequencing)、末端標結'碟酸略定 序（terminally-labeled phosphate sequencing)與擺動定序 (wobble sequencing)。在其他實施例中，定序反應為一驗基 延伸定序反應。在其他實施例中，鹼基延伸定序反應更包 括將經阻礙(blocked)與經標誌之核苷酸加至光學偵測裝置 的步驟。在其他實施例中，核苷酸為經螢光標誌的。 1378237 在另一方面，實施例也提供偵測一樣本分子的方法。 在一些貫施例中，這些方法包括將經標誌之樣本附著至本 發明所提供之光學偵測裝置上的連結位及在—對應之光偵 測器上偵測樣本分子的步驟。在一些實施例中，樣本分子 藉由連結分子附著至一連結位。在一些實施例中，連結 分子包括1) 一適合與樣本分子結合之捕獲分子（capture molecule)與2) —核酸標籤。在其他實施例中，將樣本分 子提供於例所提供之連結分子已附著於連結位上的光6. Description of the Invention: [Technical Field] The present invention relates to a bioanalytical system including a plurality of optical detecting devices and a bioanalytical system for detecting and analyzing biomolecules, and biomolecules such as nucleic acids . In one embodiment, the invention relates to a bioanalytical system comprising at least 10,000 optical detection devices for monitoring, in some embodiments, a plurality of fluorophore molecules for parallel detection Measuring and analyzing biomolecules. [Prior Art] The Human Genome Project (HGP) stimulates a large increase in the amount of sequencing processing and leads to a relatively low ordering cost. Compared to the cost of 13 years and nearly $3 billion per genomic body, the cost of sequencing has been significantly reduced. To be exact, two separate genomes have recently been completed (McGuire et al., 317:1687 (2007) ). The personal genome represents a paradigm shift in medical treatment for both patients and health care providers. By managing the genetic risk factors for disease, health care providers can implement preventive drugs and provide customized treatments more immediately. Due to the completion of the large genomic banks, drug design and drug administration can be more effective, pushing the initial field of pharmacogenetics forward. In order to popularize custom-made drugs for individuals, the National Institute of Human Genetics (NIH) National Institute of Human Genetics (NhgRI) has determined that the cost of sequencing each genome has dropped from a few million to nearly a thousand dollars. However, the general two-flux capillary electrophoresis and automated genomic sequencing techniques do not meet the increased demand for individual genome sequencing. In addition, the ordering method requires complex and error-prone images 撷2 and analysis steps. For example, in order to obtain multiple images, many current techniques are either arrays or detection systems to move. The resulting images can then be arranged, aligned and analyzed sequentially. Image Capture, Processing, and Analysis Steps White tends to be erroneous, takes extra time, and requires expensive equipment. Conversely, current systems that do not include mobile optics are often limited because of the large number of pre-test units. Finally, the current component does not place the molecule to be detected close to a corresponding detection unit, which substantially limits the strength of the detection signal. Therefore, there is a need for a device that reduces the cost of nucleic acid sequencing. In order to achieve the "$1000 genomic, paradigm, components need to be able to sequence multiple molecules in parallel, have simplified design and manufacturing processes, and avoid the need for scanning and image analysis of complex and error-prone trends in existing components and methods. The element needs to have the ability to sequence a single molecule to avoid expansion of the clustered sequencing method (eg, drift between sequences of ideal replica templates) and sequencing (step-by-step sequence reaction between sequencing templates) Dephasing of the stepwise sequencing reaction. The difficulty of the known different periods in the two steps. [Invention] The present invention provides a biological analysis system including a plurality of optical detecting devices and uses for nucleic acid. A method of detecting a biological analysis system, such as sequencing. The bioanalytical system provided in the examples has the capability of a large-scale parallel sequencing reaction, 1378237, ie, a large number of different nucleic acid templates are simultaneously performed. Sequencing. Each sequencing reaction makes - using a single molecule as a template (ie 'single molecule sequencing') The provided components and components also have a simplified design - eliminating the need for complex, expensive = error-prone scan and detection steps of the current components. The simplified design and system functionality provided by the present invention is based in part on To be directly associated with the linker site of the [detected nucleic acid (not directly, for example, by a polymerase molecule) with one or more detection units (eg. , the light detector), and based in part on the short distance between the link and the detection unit. In some embodiments, the large solid angle (s〇Hd φ angle) is displayed. The short distance between the nucleic acid and the detection unit. In one aspect, a biometric system for identifying a single biomolecule in a detection unit is provided. The bioanalysis system can include a plurality of optical detection devices. a substrate having a photodetector, the substrate being configured to detect light emitted from biomolecules. In some embodiments, the photodetector can include a charge-coupled device, a complementary metal oxygen One of a semiconductor (compoundary metal-oxide semiconductor) sensor or a photodiode. The optical detecting device can include a blind sheet on the substrate, and the masking sheet includes A pinhole, and a filter layer are provided between the masking sheet and the substrate and are provided below the pinhole, the filter layer being non-planar and arranged to filter light emitted from the biomolecule. In some embodiments, portions of the non-planar filter layer can be arcuate, spherical, elliptical or arcuate. The optical detection device also includes a "coupling position" which is provided close to the pinhole, and the bonding site is processed to place the biomolecule close to the pinhole. In another aspect, a bioanalytical system for identifying a single biota [s] 6 1378237 : sub is provided in a detection unit. The bioanalytical system can include a plurality of optical detection devices, each of which can include a substrate having a photodetector, the slab being configured to detect light emanating from the biomolecule. The optical detecting device can include a light emitting layer formed on the substrate, wherein the light emitting layer includes a recess, and a filter layer is provided between the recess and the substrate and under a pinhole. The layers are non-planar and are arranged to filter light that is emitted from biomolecules. In some embodiments, portions of the non-planar filter layer may be arcuate, spherical, elliptical or arcuate. The optical detection device can also include a nnker site, Φ being provided proximate to the recess and the pinhole, the bonding site being processed to place the biomolecules close to the pinhole. The optical detection device can further include an excitation light source to provide a light source for exciting a fluorophore attached to the biomolecule. In another aspect, another embodiment provides a link by attaching at least - a nucleic acid to an optical detection device of an embodiment (not directly, by binding to a nucleic acid polymerase that binds to a linker) And detecting a nucleic acid detecting nucleic acid on a corresponding photodetector. In some embodiments, the detection nucleic acid is hybridized with, for example, a labeled probe. In some embodiments, the nucleic acid is detected by performing nucleic acid sequencing on the optical detection device. In some embodiments, the nucleic acid sequencing method is selected from base-extension sequencing, terminally-labeled phosphate sequencing, and wobble sequencing. In other embodiments, the sequencing reaction is an experimental extension of the sequencing reaction. In other embodiments, the base extension sequencing reaction further includes the step of adding blocked and labeled nucleotides to the optical detection device. In other embodiments, the nucleotides are fluorescently labeled. 1378237 In another aspect, the embodiments also provide methods of detecting the same molecule. In some embodiments, the methods include the step of attaching the marked sample to a link on an optical detection device provided by the present invention and detecting a sample molecule on a corresponding photodetector. In some embodiments, the sample molecules are attached to a linking site by a linking molecule. In some embodiments, the linker molecule comprises 1) a capture molecule suitable for binding to the sample molecule and 2) a nucleic acid tag. In other embodiments, the sample molecules are provided with light that has been attached to the linking sites by the linking molecules provided by the examples.

學_裝置。在其他實_中’允許—樣本分子與一連結 分子結合’且之後將結合複合物提供於光學制裝置並允 許其附著至—連結位。在其他實施例t，樣本分子為-生 物分子，例如一多胜肽、核酸、脂質、多醣或代謝物 (metabolite)。 為了讓本發明之上述和其他目的、特徵、和優點能更 明顯易懂，下文特舉較佳實施例，並配合所附圖示，作詳 細說明如下： 【實施方式】 1.生物分析系統 生物分析系統可用來平行監測大量（例如，在一些實 施例中，大於1G，_)之單-分子。生物分析系統可包括 複數個光學㈣裝置。各光學勤m置可檢測出於單一分 子上之營光_u⑽Ph〇re)的存在，藉由_散發自營光團 的光子。藉由平行齡光學❹禮置，生物分析系統可以 1378237 乂 高通量確認，例如一基因體之序列或於一組織樣本中被表 現之基因的輪靡(profile)。 參見第1圖，顯示一生物分析系統1。生物分析系統1 可包括生物分析基板10與複數個光學偵測裝置20形成於 基板10上。可獨立操作各光學偵測裝置20以偵測並鑑定 附著於其之一單一生物分子。例如，藉由連續執行一驗基 • 延伸與使用光學偵測裝置20偵測散發自與經延伸之鹼基 ' 結合之一螢光團的光可確認一單股DNA之序列。藉由將大 • 量之光學偵測裝置20整合於基板10上，可平行偵測並確 認大量之單一生物分子。根據設計選擇，生物分析系統1 可包括至少，例如一萬（1〇,〇〇〇)、二十五萬（250,000)、二百 萬（2,000,000)、或甚至一千萬（10,000,000)或更多個光學偵 測裝置20形成於基板10上。 生物分析系統1可更包括一與基板10耦合之偵測與記 錄系統2，用以控制光學偵測裝置20的操作與用以記錄擷 取自光學偵測裝置20的資料。此外，生物分析系統1可更 ® 包括一激發光源（未顯示）。激發光源可產生激發光，以 便誘導螢光團散發螢光。在一實施例中，激發光源可離開 光學偵測裝置20或生物分析基板10單獨設置。在替代實 施例中，可將激發光源與光學偵測裝置20或生物分析基板 10進行整合。 於此實施例中，如第1圖中所示，當自上方觀看時， 光學偵測裝置20可具有一圓形的形狀。可以瞭解的是，光 學偵測裝置20可具有其他幾何形狀，例如一正方形的形 狀、一多邊形的形狀、一卵形的形狀與其類似。另外，第 1378237 1圖顯示複數個光學偵測裝置20被配置於一方格圖案中。 可以瞭解的是，光學偵測裝置20可被配置於其他圖案中， 例如三角格圖案、一蜂巢格圖案與其類似。 由於生物分析系統1之複數個光學偵測裝置20為可獨 立地操作，將於以下依照各種實施例描述要被使用之一光 學偵測裝置20。雖然將描述一光學偵測裝置，但可理解 的疋，於生物分析系統1中不同之光學偵測裝置2〇沒有必· 要相同。根據设計選擇，根據不同實施例可建構不同形式 之光學偵測裝置20。 參見第2圖’其顯示沿著第1圖之線a_a，依照一實 施例之光學偵測裴置20的一剖面圖。如第2圖中所示，光 學偵測裝置20包括一光偵測器21〇形成於基板1〇上與一 連結位220形成於光偵測器21〇上。此外，光學偵測裝置 20可更包括一控制電路215形成於基板1Q上用以控制 光感測器210之操作。控制電路215可與偵測與記錄系統 2耦合以便接受來自偵測與記錄系統2之控制指示並傳送 經偵測之訊號至偵測與記錄系統2。在一些實施例中，基 板10可為一玻璃基板、一半導體基板（例如，矽）或一塑 膠基板。在一些實施例中，一或更多之控制電路215可相 當於各光偵測器210。 在一些實施例中’光偵測器210可包括一單一光電導 (photoconductive)光子偵測器或一組之光電導光子偵測 器’例如一或更多之電何编合元件（charged-coupled device) 或互補金屬氧化半導體（complementary metal-oxide semiconductor，CMOS)感測器。在替代實施例中，光偵測器m 1378237 .- 210可包括一單一光電壓（photovoltaic)光子偵測器或一組 ^ 之光電壓光子偵測器。在替代實施例中，光偵測器210可 包括一單一累崩光二極體（avalanche photodiode)或一組之 累崩光二極體。在替代實施例中，光偵測器210可包括一 單一光電晶體(phototransistor)或一組之光電晶體。 . 在一實施例中，光學偵測裝置20可更包括一遮蔽薄板 .(blind sheet)230於光偵測器210上。遮蔽薄板230可包括 一針孔235。在一實施例中，針孔235可具有一圓形之形 % 狀且可具有小於或等於10,000、1，000、500、300、200、 150或100 nm之一直徑D1。可以理解的是，針孔235可 具有其他形狀，例如一卵形之形狀、一正方形之形狀與其 類似。在一實施例中，遮蔽薄板230可包括一不透光材料， 以便阻擋不期望之光到達光偵測器210。因此，所期望之 光線可經由針孔235到達光偵測器210。 可將連結位（linker site)220形成接近於針孔235。於顯 示在此圖中之實施例中’連結位220形成於針孔235之内。 • 在一實施例中，藉由一小於或等於100 μιη之距離Η1可將 - 形成接近於針孔235之連結位220與光偵測器210隔開。 在替代實施例中，距離Η1可小於或等於75、50、25、15、 10、5 或 3 μπι。 光學偵測裝置20可更包括一濾層240 (光學）與一微 透鏡(microlens)250 (光學）介於光偵測器210與遮蔽薄板 230之間。雖然第2圖顯示濾層240係形成在微透鏡250 上，可以理解的是’濾層240可形成在微透鏡25〇之下。 在一些實施例中，濾層240可包括一單一透明層或複數個 1 11 1378237 具有不同折射率(_aetive index)之透明次層㈣灿㈣。 如於第3a圖中所示，於一些實施例中，複數個次層可 為非平面非平面可包括’但不限於，半球形、橢圓形、 弓形或除此H線形之形狀及/或表面。當制24〇包括 複數個弧線形次層時，藉由連續沈積次層於基板上可形 成滤層24G、。於操作巾，複數個弧_次層可具有光學傳 送特f生其為-特定波長或波長範圍之高度地反射及有關 於一不同波長或波長範圍之逆向地傳達。例如，於一實施 例中之複數個弧線形濾膜可反射波長633 nm之光，而發射 具有680 nm之波長的光。在一些實施例中，可將一具有較 高折射率之次層失在具有較低之折射率之兩次層之間。或 者’可將一具有較低折射率之次層夹在具有較高之折射率 之兩次層之間。在一些實施例中，濾層240可包括具有單 一區域的一層或具有複數個次區域的一層，而複數個次區 域具有對不同波長範圍不同之透明度。 m 回到第2圖’可對連結位22〇進行處理以使生物分子 30附著於其。在一實施例中，生物分子30可包括一單股 DNA分子32及與單股dna分子32結合之一末端引子（end primer)34。藉由末端連結引子34，生物分子3〇可附著於 連結位220。再者，可以一螢光團36來標誌DNA分子32。 當被一第一波長λΐ之激發光激發時，螢光團36可散發一 第二波長λ2之螢光。在一些實施例中，第一波長λΐ比第 二波長λ2短。在一些實施例中，第一波長χι比第二波長 λ2長’例如於多光子激發(mUiti_ph〇ton excitation)中。之後 光偵測器210偵測散發自螢光團36之螢光以便鑑定螢光團 12 1378237 36所附著之鹼基形式’藉此連續地測定DNA分子32的序 列。 參見第3b圖，其顯示根據一實施例之光學偵測裝置 20的一剖面圖。如於第3b圖中所示，遮蔽薄板230形成 於光價測器210之上，且藉由距離H1與光感測器210垂 直地間隔。具有厚度T之遮蔽薄板23〇包括具有半徑 (即，直徑D1之一半）之針孔235。於此實施例中，連結 位220可形成於針孔235中以與一生物分子（未顯示）結 合。 當螢光團36設置在位於連結位220上之一第一位置 36A且藉由一距離H2與連結位220分開時，具有半徑R2 之光偵’則器21 〇可在一第一立體角（犯仙angie)0丨内收集散 發自螢光團36的螢光。當螢光團36設置在一第二位置36B 且幾乎與連結位22〇接觸（即，距離H2接近零或小於1 μιη) 時’光偵測器210之後可在一第二立體角Θ2内收集散發自 螢光團36的螢光。第二立體角θ2大於第一立體角Θ1且提 供一實質上較強的訊號。 為了使光偵測器210經由針孔235暴露於散發自螢光 團36之螢光，光偵測器21〇之半徑R2必須大於或等於相 虽於投射在光偵測器21〇上表面上的第二立體角θ2的半 徑。藉由使遮蔽薄板230 (或連結位220)更接近光偵測器 210 (即，藉由減少距離Η1)，光偵測器之後可自一立體 角内收集更集中之光（即，—較強之光訊號）。在一實施 例中，藉由一'!、距離Η1來將遮蔽薄板230(或連結位220) 與光偵測器210分開’以使第二立體角θ2為至少〇 8SI球 [S} 13 1378237 面度（steradian)。 參見第4圖，其顯示沿著第1圖之線A-A，依照另一 實施例之光學偵測裝置20的一剖面圖。於此實施例中，將 一激發光源40與光學偵測裝置20進行整合。如第4圖中 所示，激發光源40形成於光學偵測裝置20之遮蔽薄板230 之上。在一實施例中，激發光源40可包括一 p型與一 η型 半導體層（410與430)，及一光散發層420介於層410與 層430間的接面區之間。層410與層430可連接至一電源。 根據用於層410、420與430之材料及/或材料之物理與原 子結構，激發光源40可為一發光二極體(light emitting diode， LED)、一發光雷射二極體(light emitting laser diode，LD)、 一有機發光二極體（organic light emitting diode，OLED)或一 高分子發光二極體(polymer light emitting diode，PLED)。無 機材料’例如、砷化鏍、磷化銦、銻化鎵與氮化鎵，或有 機材料’例如具有聚對苯乙烯 poly(para-pheneylene-vinylene)骨架之共軛高分子 (conjugated polymer)，皆為可用來製造散發光之接面二極 體(junction diode)之半導體材料的例子。 在其他實施例中’激發光源40可形成遮蔽薄板230或 可被形成於遮敗薄板230之内。在一些實施例中，與光學 偵測裝置20整合之激發光源4〇可散發一波長帶或複數個 波長帶的光。激發光源40可間歇或持續地散發光。激發光 源40可一次散發一波長帶的光或者同時散發數個波長帶 的光。 再次參見第4圖，激發光源4〇可包括一凹處450於其 14 1378237 \ 一中央部分以便露出針孔235。在此實施例中，連結位220 " 可不形成於針孔235中。當然，連結位220可形成於凹處 450中且接近於針孔235。在實施例中，其中激發光源40 形成遮蔽薄板230或被形成於遮蔽薄板230之内’凹處450 形成針孔235或被形成於針孔235之内。在一些實施例中’ 針孔235可形成在層410與遮蔽薄板230兩者之一中央部 分，藉由，例如，使用適合的製程來蝕刻層410與遮蔽薄 • 板 230。 馨 此外，經由一形成於下層410上之金屬接觸415與一 形成於上層430上之金屬接觸435，一激發光源40可與一 電源440耦合。電源440可單獨設置且藉由偵測與記錄系 統2來控制，或可與债測與記錄系統2整合。 激發光源4〇之光散發層420可散發激發光沿著一水平 方向進入凹處45〇，而水平方如於第4圖中繪於光散發層 、，^之茹項所指示。在此實施例中，激發光沿著貫男上 鲁^亍於遮蔽板230之-上表面的方向散發。因此，激發 :可以不干涉到達光偵測H 21G之g光。光學偵測裝置因 可比^又凡件更準確地鑑定生物分子。 2.核酸偵蜊 可使用生物分析系統（包括’例如不是單〆光學偵、測 :置：是複數個此種裝 置）成為一部份系統用在分子憤測 ^。刀子偵喇之方法或製程中，而分子偵測，例如核酸定 偽此生物分析系統及利用其之方法與製程對於’例如分 〃 β斷應用為有用的。這些應用可為個人的、公眾的、[ 15 1378237 商業的或工業的。 在一些實施例中’生物分析系統適合核酸之大量平行 定序。部分由於連結位與生物分析系統之光偵測器的直接 聯繫及/或連結位與光偵測器的緊密接近（在一些實施例 中’顯示為一大的立體角），可使用實施例中所提供之生 物分析系統來對核酸進行定序，而不需昂貴、複雜與錯誤 傾向之知0¾與刀析系統，例如一移動掃猫透鏡（m〇ving scanning lens)或一移動元件台⑼⑽吨device stage)與隨後 的影像分析’因此減少錯誤與成本。生物分析系統可偵測 具有實質上經改善之訊號強度的訊號，其使單一分子分析 變成可能。 可以廣泛變化之定序形式來使用生物分析系統，且生 $分析系統適合將單一分子進行定序。另外，與現行生物 二片7L件相較’光學偵測元件具有經簡化之設計、裝配及 °例如’可將要被定序之核酸附著於陣列上之隨機的 ’避免對於在預定位置沈積或合成核酸之時間消耗 與昂責機器的使用。 巾可使用生物分析系統為在生物分子偵 測之方法與程序 體t 系統的部分，包括核酸雜合或對於，例如整個基因 序之疋序、轉錄表現(transcriptional profiling)、競爭轉 錄表現或基因鑑定。生物分子偵測也可包括結合相互作 白’列如蛋白質/蛋白質、抗體/抗原、受器/配體及核酸/蛋 、質之侦測及/或測量。這些應用對於分析與診斷程序與方 法為有用的。Learn _ device. In other instances, the sample molecule is allowed to bind to a linker molecule and the bond complex is then provided to the optical device and allowed to adhere to the link site. In other embodiments t, the sample molecule is a biomolecule such as a multi-peptide, nucleic acid, lipid, polysaccharide or metabolite. The above and other objects, features, and advantages of the present invention will become more apparent from the description of the preferred embodiments illustrated herein The analytical system can be used to monitor a large number (e.g., in some embodiments, greater than 1G, _) of single-molecules in parallel. The bioanalysis system can include a plurality of optical (four) devices. Each optical device can detect the presence of camp light _u(10)Ph〇re) on a single molecule, and emit photons of self-operated light groups by _. With parallel-aged optics, the bioanalytical system can be identified by high-throughput 1378237 ,, such as a sequence of genes or a profile of genes expressed in a tissue sample. Referring to Figure 1, a bioanalysis system 1 is shown. The bioanalysis system 1 may include a bioanalysis substrate 10 and a plurality of optical detecting devices 20 formed on the substrate 10. Each optical detection device 20 can be independently operated to detect and identify a single biomolecule attached to it. For example, the sequence of a single strand of DNA can be confirmed by continuously performing a test. • Extension and detection of light emitted from a fluorescent group bound to the extended base by the optical detecting device 20. By integrating a large number of optical detecting devices 20 on the substrate 10, a large number of single biomolecules can be detected and confirmed in parallel. Depending on the design choice, the bioanalysis system 1 may include at least, for example, 10,000 (1, 〇〇〇), 250,000 (250,000), 2 million (2,000,000), or even 10 million (10,000,000) or more. An optical detecting device 20 is formed on the substrate 10. The biometric system 1 can further include a detection and recording system 2 coupled to the substrate 10 for controlling the operation of the optical detection device 20 and for recording data retrieved from the optical detection device 20. In addition, the bioanalysis system 1 can include an excitation source (not shown). The excitation source produces excitation light to induce the fluorophore to emit fluorescence. In one embodiment, the excitation source can be separately disposed from the optical detection device 20 or the bioanalytical substrate 10. In an alternate embodiment, the excitation source can be integrated with optical detection device 20 or bioanalytical substrate 10. In this embodiment, as shown in FIG. 1, the optical detecting device 20 may have a circular shape when viewed from above. It will be appreciated that the optical detection device 20 can have other geometric shapes, such as a square shape, a polygonal shape, an oval shape, and the like. Further, reference numeral 1378237 1 shows that a plurality of optical detecting devices 20 are arranged in a one-frame pattern. It can be appreciated that the optical detection device 20 can be configured in other patterns, such as a triangular lattice pattern, a honeycomb pattern, and the like. Since the plurality of optical detecting devices 20 of the bioanalytical system 1 are independently operable, one of the optical detecting devices 20 to be used will be described below in accordance with various embodiments. Although an optical detecting device will be described, it will be understood that the different optical detecting devices 2 in the biological analysis system 1 are not necessarily the same. Depending on the design choice, different forms of optical detection device 20 can be constructed in accordance with various embodiments. Referring to Fig. 2', a cross-sectional view of the optical detecting device 20 in accordance with an embodiment is shown along line a_a of Fig. 1. As shown in FIG. 2, the optical detecting device 20 includes a photodetector 21 formed on the substrate 1 and a connecting portion 220 formed on the photodetector 21A. In addition, the optical detecting device 20 may further include a control circuit 215 formed on the substrate 1Q for controlling the operation of the photo sensor 210. Control circuitry 215 can be coupled to detection and recording system 2 for accepting control indications from detection and recording system 2 and transmitting the detected signals to detection and recording system 2. In some embodiments, the substrate 10 can be a glass substrate, a semiconductor substrate (e.g., germanium), or a plastic substrate. In some embodiments, one or more control circuits 215 can be appropriate for each photodetector 210. In some embodiments, 'photodetector 210 can include a single photoconductive photon detector or a set of photoconductive photodetectors' such as one or more electrically coupled components (charged-coupled) Device) or a complementary metal-oxide semiconductor (CMOS) sensor. In an alternate embodiment, photodetector m 1378237 .-210 may include a single photon photon detector or a set of photovoltage photon detectors. In an alternate embodiment, photodetector 210 can include a single avalanche photodiode or a set of discrete photodiodes. In an alternate embodiment, photodetector 210 can comprise a single phototransistor or a group of optoelectronic crystals. In an embodiment, the optical detecting device 20 further includes a blind sheet 230 on the photodetector 210. The masking sheet 230 can include a pinhole 235. In an embodiment, the pinhole 235 can have a circular shape and can have a diameter D1 that is less than or equal to one of 10,000, 1,000, 500, 300, 200, 150, or 100 nm. It will be appreciated that the pinhole 235 can have other shapes, such as an oval shape, a square shape, and the like. In an embodiment, the masking sheet 230 can include an opaque material to block unwanted light from reaching the photodetector 210. Therefore, the desired light can reach the photodetector 210 via the pinhole 235. A linker site 220 can be formed proximate to the pinhole 235. In the embodiment shown in this figure, the joint position 220 is formed within the pinhole 235. • In one embodiment, the junction 220 formed to be close to the pinhole 235 can be separated from the photodetector 210 by a distance Η1 less than or equal to 100 μm. In an alternate embodiment, the distance Η1 can be less than or equal to 75, 50, 25, 15, 10, 5, or 3 μπι. The optical detecting device 20 may further include a filter layer 240 (optical) and a microlens 250 (optical) interposed between the photodetector 210 and the shielding sheet 230. Although FIG. 2 shows that the filter layer 240 is formed on the microlens 250, it is understood that the 'filter layer 240 may be formed under the microlens 25'. In some embodiments, the filter layer 240 can comprise a single transparent layer or a plurality of transparent layers (4) of a different refractive index (4). As shown in FIG. 3a, in some embodiments, the plurality of sub-layers may be non-planar non-planar may include, but is not limited to, hemispherical, elliptical, arcuate or otherwise H-shaped shapes and/or surfaces. . When the 24 〇 includes a plurality of arcuate sublayers, the filter layer 24G can be formed by continuously depositing the sublayer on the substrate. In the operation of the towel, the plurality of arc-sublayers may have optical transmissions that are - highly reflective of a particular wavelength or range of wavelengths and are inversely related to a different wavelength or range of wavelengths. For example, a plurality of arcuate filters in an embodiment can reflect light having a wavelength of 633 nm and emit light having a wavelength of 680 nm. In some embodiments, a sub-layer having a higher refractive index can be lost between two layers having a lower refractive index. Alternatively, a sublayer having a lower refractive index may be sandwiched between two layers having a higher refractive index. In some embodiments, filter layer 240 can include a layer having a single region or a layer having a plurality of sub-regions, while the plurality of sub-regions have different transparency for different wavelength ranges. m Returning to Figure 2, the link 22 can be treated to attach the biomolecule 30 thereto. In one embodiment, biomolecule 30 can include a single strand of DNA molecule 32 and an end primer 34 in combination with a single strand of DNA molecule 32. The biomolecule 3 can be attached to the linking site 220 by the terminal linking primer 34. Further, a fluorescent cluster 36 can be used to mark the DNA molecule 32. When excited by the excitation light of a first wavelength λ , , the fluorophore 36 can emit fluorescence of a second wavelength λ 2 . In some embodiments, the first wavelength λ 短 is shorter than the second wavelength λ 2 . In some embodiments, the first wavelength χι is longer than the second wavelength λ2, e.g., in a multi-photon excitation (mUiti_ph〇ton). The photodetector 210 then detects the fluorescence emitted from the phosphor group 36 to identify the base form to which the fluorophore 12 1378237 36 is attached' thereby thereby continuously determining the sequence of the DNA molecule 32. Referring to Figure 3b, a cross-sectional view of optical detection device 20 in accordance with an embodiment is shown. As shown in Fig. 3b, the masking sheet 230 is formed on the photometric detector 210 and is vertically spaced from the photosensor 210 by a distance H1. The masking sheet 23 having a thickness T includes a pinhole 235 having a radius (i.e., one half of the diameter D1). In this embodiment, a bonding site 220 can be formed in the pinhole 235 to bond with a biomolecule (not shown). When the luminescent group 36 is disposed at one of the first positions 36A at the joint position 220 and separated from the joint position 220 by a distance H2, the ray detector 21 having the radius R2 may be at a first solid angle ( In the 仙 ang ang ) ) 收集 收集 收集 收集 收集 收集 收集 收集 收集 收集 收集 收集 收集 收集 收集When the luminescent group 36 is disposed at a second position 36B and is almost in contact with the bonding position 22 (ie, the distance H2 is near zero or less than 1 μm), the photodetector 210 can be collected in a second solid angle Θ2. Fluorescence emitted from the fluorescent group 36. The second solid angle θ2 is greater than the first solid angle Θ1 and provides a substantially stronger signal. In order to expose the photodetector 210 to the fluorescent light emitted from the fluorescent group 36 via the pinhole 235, the radius R2 of the photodetector 21 must be greater than or equal to that projected on the upper surface of the photodetector 21 The radius of the second solid angle θ2. By bringing the masking sheet 230 (or the joining position 220) closer to the photodetector 210 (ie, by reducing the distance Η1), the photodetector can then collect more concentrated light from a solid angle (ie, Strong light signal). In one embodiment, the masking sheet 230 (or the joint location 220) is separated from the photodetector 210 by a '! distance Η1' such that the second solid angle θ2 is at least 〇8SI sphere [S} 13 1378237 Steradian. Referring to Fig. 4, there is shown a cross-sectional view of optical detecting device 20 in accordance with another embodiment along line A-A of Fig. 1. In this embodiment, an excitation source 40 is integrated with the optical detection device 20. As shown in Fig. 4, the excitation light source 40 is formed on the masking sheet 230 of the optical detecting device 20. In one embodiment, the excitation source 40 can include a p-type and an n-type semiconductor layer (410 and 430), and a light-emitting layer 420 is interposed between the junction regions between the layer 410 and the layer 430. Layer 410 and layer 430 can be connected to a power source. Depending on the physical and atomic structure of the materials and/or materials used for layers 410, 420, and 430, excitation light source 40 can be a light emitting diode (LED), a light emitting laser (light emitting laser). Diode, LD), an organic light emitting diode (OLED) or a polymer light emitting diode (PLED). Inorganic materials 'for example, arsenic arsenide, indium phosphide, gallium antimonide and gallium nitride, or organic materials' such as a conjugated polymer having a poly-para-pheneylene-vinylene skeleton, They are all examples of semiconductor materials that can be used to fabricate junction junction diodes. In other embodiments, the excitation source 40 can form a masking sheet 230 or can be formed within the masking sheet 230. In some embodiments, the excitation source 4A integrated with the optical detection device 20 can emit light in a wavelength band or a plurality of wavelength bands. The excitation light source 40 can emit light intermittently or continuously. The excitation light source 40 can emit light of one wavelength band at a time or simultaneously emit light of several wavelength bands. Referring again to Figure 4, the excitation source 4A can include a recess 450 in its central portion to expose the pinhole 235. In this embodiment, the joint position 220 " may not be formed in the pinhole 235. Of course, the joint location 220 can be formed in the recess 450 and proximate to the pinhole 235. In an embodiment, wherein the excitation source 40 forms a masking sheet 230 or is formed within the masking sheet 230, the recess 450 forms a pinhole 235 or is formed within the pinhole 235. In some embodiments, the pinholes 235 can be formed in a central portion of both the layer 410 and the masking sheet 230 by etching the layer 410 and the masking sheet 230, for example, using a suitable process. In addition, an excitation source 40 can be coupled to a power source 440 via a metal contact 415 formed on the lower layer 410 and a metal contact 435 formed on the upper layer 430. The power source 440 can be separately provided and controlled by the detection and recording system 2, or can be integrated with the debt measurement and recording system 2. The light emitting layer 420 of the excitation light source 4 emits excitation light into the concave portion 45 沿着 in a horizontal direction, and the horizontal side is as indicated in Fig. 4 on the light emitting layer. In this embodiment, the excitation light is emitted in the direction of the upper surface of the shield plate 230. Therefore, the excitation: can not interfere with the light reaching the light detecting H 21G. Optical detection devices are more accurate in identifying biomolecules than can be used. 2. Nucleic Acid Detection A bioanalytical system (including, for example, not a single optical detection, measurement: a plurality of such devices) can be used as part of the system for molecular inversion. In the method or process of knife detection, molecular detection, such as nucleic acid sequencing, and the methods and processes utilizing the same are useful for, for example, splitting. These applications can be for personal, public, [ 15 1378237 commercial or industrial. In some embodiments the 'biological analysis system is suitable for a large number of parallel sequencing of nucleic acids. In part, due to the direct connection of the link to the photodetector of the bioanalytical system and/or the close proximity of the link to the photodetector (shown as a large solid angle in some embodiments), embodiments may be used A bioanalytical system is provided to sequence nucleic acids without the need for expensive, complicated, and error-prone knowledge, such as a m〇ving scanning lens or a moving component (9) (10) ton Device stage) and subsequent image analysis 'thus reducing errors and costs. Bioanalytical systems detect signals with substantially improved signal intensities that make single molecule analysis possible. Bioanalytical systems can be used in a widely varying sequence, and the bioanalytical system is suitable for sequencing a single molecule. In addition, compared to current bio-two-piece 7L devices, the optical detection element has a simplified design, assembly, and, for example, randomization of the nucleic acid to be sequenced onto the array to avoid deposition or synthesis at predetermined locations. The time consumption of nucleic acids and the use of the machine. The tissue can be used in bioassay systems for both biomolecule detection methods and portions of the program t system, including nucleic acid hybridization or for, for example, whole gene sequence sequencing, transcriptional profiling, competitive transcriptional expression, or genetic identification. . Biomolecular detection can also include detection and/or measurement of binding to each other as a protein such as protein/protein, antibody/antigen, receptor/ligand, and nucleic acid/egg. These applications are useful for analysis and diagnostic procedures and methods.

適合於本發明所提供之系統上偵測之核酸，於一些實iSSuitable for detecting nucleic acids on the system provided by the present invention, in some real iS

施例中，為連結八2 A 互作用之分子，：=:，，其將一適合於分析結合相 或小分子附著 蛋白貝、其他減、碳水化合物部分 . 〜實施例所提供之元件上的連結位。在一 ;、她例中’連結分子可更包括—捕獲分子其由於結合 酸，=用與要破分析之分子結合。在—連結分子中之核 子的二定::直接定序或雜合’作為連結分子之捕獲分 ^貫彳〗斤提供之方法包括將要被偵測之分子附著於實 =例所提供之疋件的一位址陣列（address array)的一步驟。 在一些實施例中 ’位址陣列可包括具有複數個針孔235之 板230，與可形成在針孔235中或環繞針孔235之 叮。參見，例如第1與2圖。因此生物分析系統 。5 °貝取數百萬之核酸片段。若各片段為，例如1000鹼 基長，單—— 疋件可獲得數十億位元(bit)之序列資料，而 使整個基因體之定序與再定序（resequendng)為可能。 2.1要被偵蜊之分子 適合以實施例所提供之方法來偵測之核酸可包括任何 才只酉夂 0括例如 DNA、RNA 或 PNA (peptide nucleic acid (胜月太核酸））’且可含有任何序列-已知與未知包括自 序列。核酸可被自然取得、重組產生或化學 合成。核酸可包括自然發生㈣酸、不存在於自，然之核苷 酸類似物或經修飾之核苷酸。基於實際應用，被偵測之核 酸長度可多樣化。在一些實施例中，核酸包括至少10、20、 50、100、200、500、1000、2000、5000、10000、20000 ί S 1 17 1378237 個驗基或更多。在一些實施例中，核酸可為自10至20、 自10至50、自10至100、自50至100、自50至500、自 50 至 1000、自 50 至 5000、自 500 至 2000、自 500 至 5000， 或自1000至5000個鹼基。 用以偵測之核酸可為單股。單股核酸模板可來自一雙 股核酸模板，藉由本技術領域所知的方法，例如加熱或鹼 或其他化學處理。藉由例如化學—^，請·⑽合成，也可產生 單股核酸模板。 在一些實施例中，要被相之㈣於其5，或3，端附著 -連結位。在-些實_中，_可更包括—或多個與核 酸之5’端、3’端或5’端與3’端㈣結合之末端連結引子。 在其他實施例中’-末端連結弓丨子附著至核酸之3,端。可 Γ士末端子來將要被偵剛之核酸附著於在元件上之 =位=Γ對於—或多個偵測之引子之互補序列， 例如一定序引子。 2.1.1末端連結5|子 末端：結二：為短的核酸分子，通常由少請個核 二二i5、2〇、t貫施例中’末端連結引子之長度為至 ^一 ' 30 ' 50、75、90個核苷酸或更多。 在貫施例中，末端連則子之長 & 20、自10至30，或自1〇至 日υ芏 中，末端連結引子為未經分 其可為經分支的。 的’然而’在其他實施例中， 可使用末端連結引子來將要被俄測之核酸附著至於位 18 1378237 置陣列上之連結位。在一些實施例中’末端連結引子可將 核酸直接地連結至陣列表面，例如藉由共價連結（例如酉旨 (ester)或硫醇(thiol)連結）或非共價連結’例如抗原/抗體咬 生物素(biotin)/抗生物素蛋白（avidin)結合。參見，例如第6 圖、第7圖與第8圖。在一些實施例中’末端連結弓丨子可 將核酸非直接地連結至陣列表面，例如藉由與一居中分子 (intermediate molecule)結合，而居中分子，例如一聚合酶。 . 參見，例如第8圖。因此，末端連結引子可包含經修飾之 φ 核酸或以其他方式修飾以協助附著至一連結位，藉由本技 術領域已知的方法，例如，雙硫、硫酯、醯胺、磷酸二酯 或酯連結；或藉由，例如抗體/抗原，或生物素/抗生物素蛋 白結合，例如末端連結引子包含—包括一抗原區域之核苷 酸或一經生物素化（biotinylated)之核苷酸。在其他實施例 中，一經修飾之核苷酸位於一末端連結引子之3,端上。在 一些實施例中，一末端連結引子之5,端包含一經修飾之核 皆酸。 ® 末端連結引子也可做為一用以偵測核酸之一或多個引 子的互補物（complement)，例如，—定序序列。在一些實施 例中，引子係猎由雜合用來{貞測核酸，例如，引子包含一 可偵測之標誌’例如一螢光或放射同位素標德。在一些實 施例中’末端連結引子的5’端包括與一定序引子互補之序 列。在一些實施例中，與定序引子互補之末端連結弓丨子係 被定位以使定序引子之3 ’端直接與要被定序之核酸中的第 一個核苷酸鄰接。 例如’第6圖為要被定序之序列附著至一光學偵測裝 [S 1 19 1378237 置20之—實施例的圖解顯示。一單股核酸32、末端連結 與黏合定序引子（annealed sequencing primer)346 附 著於—經處理具有反應性官能基之連結位220，連結位220 與於末端連結引子34上之一經修飾之核苷酸344結合。在 些貫施例中’核酸32經由其5,端可附著至連結位220， 而末端連結引子34可附著至核酸32之3,端以作為定序引 子346之一互補物。 在些貝她例中’藉由一連接酶(ligase)，例如一 DNA 連接酶將末端連結引子加至要被偵測之核酸的末端。在一 些貝施例中’在連接（ligati〇n)前，末端連結引子與要被偵 測之核酸兩者皆為單股。在其他實施例中，兩者皆為雙股。 在另外其他實施例中，一者為單股，而另一者為雙股。連 接為本技術領域所熟知。例如，在聚合群落定序方法(p〇l〇ny sequencing method) t ，Shendure et al. (Science, 309:1728-1732(2005))以新英格蘭生物實驗室（New England Biolab，NEB)快速連接套組(Quick Ligation kit)將一 T30 末 端連結引子(32 bp)連結至一樣本DNA片段。其中，連結反 應溶液包括0.26 pMole之DNA、0.8 pMole之T30末端連 結引子、4.0 μΐ T4 DNA連接酶於lx快速連接緩衝溶液 (Quick Ligation buffer)中。於混合後，於室溫培養反應溶液 約10分鐘，且之後置於冰上。藉由將樣本加熱至65。〇，1〇 分鐘來停止連接反應。 在其他實施例中，可將末端連結引子合成於要被偵測 之核酸上。例如’末端連結引子可為一藉由例如末端轉移 酶(terminal transferase)所力σ入之均聚物（h〇mopolymer)。例 [S ] 20 1378237 如 ’ Harris etal.(5We«ce, 320:106·109(2008))將一多腺0票吟 尾巴(poly A tail)加至DNA模板，其做為於一病毒基因體 之單一分子定序中之一多胸腺嘧啶(poly T)定序引子的互 補物。 2.1.2定序引子 一定序引子為一與要被偵測之核酸的一片段或其之連 結的末端連結引子互補之単股寡核苦酸。在一些實施例 鲁 中，定序引子的長度為至少8、10、15、20、25、30、35、 40、45、50個核苷酸或更多。在特定實施例中，定序引子 之長度可為自8至25、自10至20、自10至30，或自10 至50個核苦酸。定序引子可由任何形式之核苦酸所形成， 包括自然發生核苷酸、不存在於自然之核苷酸類似物或經 修飾之核苷酸。在實施例中，在一定序引子與包括一或多 個末端連結分子之要被定序的核酸雜合後，可修飾定序引 _^子之5,端，以促進其與位置陣列上之一連結位結合。 在一些實施例中，一定序引子包含經修飾之核苷酸， ’ 例如鎖核酸(locked nucleic acid，LNA)(經修飾之核糖核甘 酸’其在一聚核酸(polynucleic acid)中提供增強之驗基堆疊 相互作用（base stacking interaction))。如鎖核酸之效用解 -t ? Levin et al. (Nucleic Acid Research 34(20):142(2006)) 顯示一含鎖核酸之引子具有經改善之專一性及顯示較強之 結合相對於對應之未鎖引子(unlocked primer)。製造mcp 1 引子（5’-cttaaattttcttgaat-3’）之三種變化’包含3個鎖核酸核 努酸（於蓋（cap)中）於引子中之不同位置： [S ] 21 1378237 MCP1 -LAN-3 ’（5 ’-cttaaattttCtTgaAt-3 ’） ； MCP1 -L AN-5 ’ (5,-CtTaAattttcttgaat-3,) ； 與 MCPl-LAN-even (5’-ctTaaatTttctTgaat-3’）。所有經鎖核酸取代之引子具有提 高之熔解溫度（melting temperature, Tm)，而 MCP1-LNA-5’ 引子顯示特別提高之定序準確度（Phred Q30 counts)。因 此，在其他實施例中，定序引子可包含至少一鎖核苷酸於 其5’端區域，即’定序引子之5’端一半、三分之一或四分 之一。 在被提供至一光學偵測元件之前，可將定序引子與單 股樣本核酸（即，一包括至少一末端連結引子之要被偵測 的核酸）雜合。可將定序引子與樣本核酸雜合，藉由將樣 本核酸與一莫耳過剩（molar excess)之定序引子混合於一含 鹽溶液中，例如 5xSSC(或 5xSSPE)、0·1% Tween20(或 0.1% SDS)與0.1% BSA緩衝溶液。可將混合物加熱65°C 至少5分鐘且緩慢冷卻至室溫以允許引子/模板黏合。藉由 適合之方法可將殘餘之引子去除，例如一分子篩（molecular sieve) 〇 {S 1 藉由適合的方法，包括序列之視覺檢閱或電腦協助之 引子設計，可設計引子，包括末端連結與定序引子兩者。 許多軟體套組為可用來協助引子設計，包括DNAStarTM (DNAstar, Inc.，Madison, WI)、OLIGO 4.0 (National Biosciences, Inc.)、Vectoe NTI®(Invitrogen)、Primer Premier 5 (Premierbiosoft)與 Primer3 (Whitehead Institute for Biomedical Research，Cambridge，MA)。引子設計，考慮到 例如，要被定序之分子、專一性、長度、所需的熔解溫度、 22 1378237 \ 二級結構、引子二聚體、GC含量、緩衝溶液的pH與離子 強度及所使用之酵素（即，聚合酶或連接酶）。參見，例 k〇 Joseph Sambrook and David Russell, Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, 3rd edition (2001)。 2.1.3結合至陣列表面 在將定序引子與包括一或多個末端連結引子之要被定 9 序的核酸黏合後，製備此複合物於一合適的緩衝溶液中、 將其挺供至·—位置陣列的表面並允許其結合。在一些實施 例中，將樣本核酸（要被偵測之核酸與一或多個末端連結 引子）附著至連結位，且之後提供定序或偵測引子。在其 他實施例中，在被提供至一元件之前，先將複合物進行雜 合。只有一個核酸樣本結合之連結位被瞭解為有效位置。 在實施例中，提供複合物至光學偵測元件且樣本核酸附著 ^ 至在位置陣列上之隨機的連結位。在其他實施例中，可提 供樣本核酸至在位置陣列上之預定的連結位，藉由適合的 ' 方法，例如藉由機器或液體處理系統。 '將核酸附著至一固體支持物之適合的方法為本技術領 域所熟知。在一些實施例中，可將樣本核酸直接附著至一 連結位，藉由共價連結，例如雙硫、硫酯、醯胺、磷酸二 S曰或酯連結；或藉由非共價連結，例如抗體/抗原，或生物 素/杬生物素蛋白結合。在一些實施例中，藉由一介於中間 的分子可將樣本核酸附著至一連結位。在一些實施例中， W於中間的分子可為一聚合酶，例如一 Dna聚合酶。 [S 1 23 1378237 如一直接、共價之核酸附著的說明例子，Adeesietal (Nucleic Acid Research，28:87 (2000))修飾一引子的 5,端以 包括一 SH g此基。根據Adeesi et al.之方法，可製備一核 酸於 50 μΜ 磷酸緩衝鹽(PBS)(NaPi: 0.1 M NaH2P04 pH 6.5， 0.1 M NaCl)中。之後可將之引子溶液提供至一經鹽 化之玻璃載片，且將其培養於一濕度控制盒於室溫中約5 小％以使引子結合至晶片表面。在結合反應完成後，以pBS 溶液於室溫震動清洗兩次，每次5分鐘，以移除未結合之 -DNA在’月/乐後，將1〇 mM β_硫氫乙醇鲁 加至一 PBS溶液且於室溫下用來沖洗位置陣列，以將未結 合之DNA的硫醇基去活化。再來，清洗陣列表面，例如一 次以5xSSC、0.1% Tween與一次以5xSSC緩衝溶液。因 此，在一些貫施例中，Adeesi et al所使用之方法可使用於 本發明所提供之方法中以將樣本核酸複合物，例如經由一 定序引子或樣本核酸之5,端附著至一連結位。 在一替代實施例中，樣本核酸可包括，例如一經生物 素化之核苷酸’且可與在連結位表面上之抗生物素蛋白結 · 合。在另一實施例中，樣本核酸可包括一抗原部分，例如 BrdU或洋地黃毒(digoxigenin)，其藉由一抗體（或其片段） 被結合於連結位上。藉由“抗體，，，可以暸解的是，此措辭 包括免疫球蛋白分子之片段，包括，例如一或多個CDR 區；或可變之重或可變之輕片段。抗體可為自然發生、重 組或合成。抗體也可包括，例如多株（P〇lyCl〇ne)與單株 (monoclone)變形。在一些實施例中，抗體以至少1〇6、1〇7、 8 9 10、10 Μ或更高之結合常數(ass〇ciati〇n constant)結合至 24 1378237 • 其抗原。抗體之結構、功能與產生為本技術領域所熟知。 . 參見’例如 Gary Howard and Matthew Kasser,In the embodiment, in order to link the octa 2 A interaction molecule, :=:, which will be suitable for analysis of the binding phase or small molecule attached protein shell, other subtraction, carbohydrate moiety. Link position. In one example, in her case, the "linking molecule may include - the capture molecule, which binds to the molecule to be broken by the binding of the acid. The determination of the nucleus in the -linked molecule: direct sequencing or heterozygous as the capture of the linked molecule. The method provided by the method includes attaching the molecule to be detected to the element provided by the actual example. A step of an address array. In some embodiments, the 'address array' can include a plate 230 having a plurality of pinholes 235, and can be formed in or around the pinholes 235. See, for example, Figures 1 and 2. Therefore the bioanalysis system. Take 5 million nucleic acid fragments at 5 °. If each fragment is, for example, 1000 bases long, a single-piece can obtain billions of bits of sequence data, and it is possible to sequence and resequend the entire genome. 2.1 The molecule to be detected is suitable for detecting the nucleic acid by the method provided in the examples, which may include any such as DNA, RNA or PNA (peptide nucleic acid) and may contain Any sequence - known and unknown includes self-sequences. Nucleic acids can be obtained naturally, recombinantly, or chemically synthesized. Nucleic acids may include naturally occurring (tetra) acids, nucleoside analogs that are not present, or modified nucleotides. The length of the nucleic acid being detected can be varied based on the actual application. In some embodiments, the nucleic acid comprises at least 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 20000 ί S 1 17 1378237 sterility or more. In some embodiments, the nucleic acid can be from 10 to 20, from 10 to 50, from 10 to 100, from 50 to 100, from 50 to 500, from 50 to 1000, from 50 to 5000, from 500 to 2000, from 500 to 5000, or from 1000 to 5000 bases. The nucleic acid used for detection can be a single strand. The single-stranded nucleic acid template can be derived from a duplex nucleic acid template by methods known in the art, such as heat or alkali or other chemical treatment. A single-stranded nucleic acid template can also be produced by, for example, chemical-^, (10) synthesis. In some embodiments, it is to be phased (4) with its 5, or 3, terminal attachment-linking sites. In the case of _, _ may further include - or a plurality of terminal linking primers which are bonded to the 5' end, the 3' end or the 5' end and the 3' end (4) of the nucleic acid. In other embodiments, the '-end linkage scorpion is attached to the 3' end of the nucleic acid. The gentleman's terminal can be used to attach the nucleic acid to be detected to the element = = Γ for - or a complementary sequence of multiple detected primers, such as a sequence of primers. 2.1.1 End-linking 5|Sub-end: Junction 2: Short nucleic acid molecule, usually from less than one nuclear two-two i5, two 〇, t-applications, the length of the end-linking primer is to ^1' 30 ' 50, 75, 90 nucleotides or more. In the examples, the length of the terminal is & 20, from 10 to 30, or from 1 to 30, the end-linking primer is undivided and can be branched. In other embodiments, end-linking primers can be used to attach the nucleic acid to be tested to the junction on the array of bits 18 1378237. In some embodiments, an 'end-linking primer can link a nucleic acid directly to the surface of the array, such as by covalent attachment (eg, ester or thiol linkage) or non-covalent linkage, eg, antigen/antibody Biotin/avidin binding. See, for example, Figure 6, Figure 7, and Figure 8. In some embodiments, the 'terminally linked tibial can bind the nucleic acid non-directly to the surface of the array, e.g., by binding to an intermediate molecule, such as a polymerase. See, for example, Figure 8. Thus, the end-linking primer may comprise a modified φ nucleic acid or otherwise modified to assist in attachment to a linker, by methods known in the art, for example, disulfide, thioester, guanamine, phosphodiester or ester. Linking; or by, for example, antibody/antigen, or biotin/avidin binding, for example, a terminal linker comprises - a nucleotide comprising an antigenic region or a biotinylated nucleotide. In other embodiments, a modified nucleotide is located on the 3' end of the terminating primer. In some embodiments, one end of the primer link 5, the end comprising a modified core acid. The ® end-linking primer can also be used as a complement to detect one or more of the nucleic acids, for example, a sequencing sequence. In some embodiments, the primer is used by hybridization to detect nucleic acids, for example, the primer comprises a detectable marker, such as a fluorescent or radioisotope marker. In some embodiments, the 5' end of the 'end joining primer includes a sequence that is complementary to a certain sequence of primers. In some embodiments, the terminal junction scorpion line complementary to the sequencing primer is positioned such that the 3' end of the sequencing primer is contiguous with the first nucleotide in the nucleic acid to be sequenced. For example, Fig. 6 is a diagram showing the sequence to be sequenced attached to an optical detecting device [S 1 19 1378237 20]. A single-stranded nucleic acid 32, an end-linked and an annealing sequence primer 346 is attached to the linker 220 having a reactive functional group, and the linker 220 is coupled to a modified nucleoside at the terminal linker 34. Acid 344 binds. In some embodiments, the nucleic acid 32 can be attached via its 5' end to the linker position 220, and the end linker 34 can be attached to the 3' end of the nucleic acid 32 to serve as a complement to the sequence leader 346. In some cases, an end-linking primer is added to the end of the nucleic acid to be detected by a ligase, such as a DNA ligase. In some of the examples, before the ligati〇n, the end-linking primer and the nucleic acid to be detected are both single-stranded. In other embodiments, both are double stranded. In still other embodiments, one is a single share and the other is a double share. Connections are well known in the art. For example, in the polymerization community sequencing method (p〇l〇ny sequencing method) t, Shendure et al. (Science, 309: 1728-1732 (2005)) quickly connected to the New England Biolab (NEB) The Quick Ligation kit links a T30 end linker (32 bp) to the same DNA fragment. Among them, the ligation reaction solution included 0.26 pMole of DNA, 0.8 pMole of T30 end ligated primer, and 4.0 μM of T4 DNA ligase in lx Quick Ligation buffer. After mixing, the reaction solution was incubated at room temperature for about 10 minutes and then placed on ice. By heating the sample to 65. 〇, 1 〇 minutes to stop the ligation reaction. In other embodiments, an end-linking primer can be synthesized on the nucleic acid to be detected. For example, the 'end-linking primer' may be a homopolymer (h〇mopolymer) which is intrinsically entangled by, for example, a terminal transferase. Example [S] 20 1378237 As in 'Harne et al. (5We«ce, 320: 106·109 (2008)), a poly A tail is added to a DNA template as a viral gene. The complement of one of the polythymidine (poly T) sequencing primers in the single molecule sequence of the body. 2.1.2 Sequencing primers A pre-sequence primer is a quinone oligo-nucleotide complementary to a fragment of a nucleic acid to be detected or a ligated end-linking primer thereof. In some embodiments, the sequencing primers are at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides or more in length. In a particular embodiment, the sequencing primers can be from 8 to 25, from 10 to 20, from 10 to 30, or from 10 to 50 nucleotides. The sequencing primer can be formed from any form of nucleotide acid, including naturally occurring nucleotides, nucleotide analogs that are not found in nature, or modified nucleotides. In an embodiment, after a certain sequence of primers is hybridized with a nucleic acid to be sequenced comprising one or more terminal linker molecules, the 5th end of the sequence can be modified to facilitate its alignment with the positional array. A joint is combined. In some embodiments, the sequence primer comprises a modified nucleotide, 'eg, locked nucleic acid (LNA) (modified ribonucleotide) that provides enhanced assays in a polynucleic acid Base stacking interaction). For example, the effect of the locked nucleic acid solution -t Levin et al. (Nucleic Acid Research 34 (20): 142 (2006)) shows that a primer containing a locked nucleic acid has improved specificity and a stronger binding relative to the corresponding Unlocked primer. Three variations of the mcp 1 primer (5'-cttaaattttcttgaat-3') were made to contain three locked nucleic acids (in the cap) at different positions in the primer: [S ] 21 1378237 MCP1 -LAN-3 '(5 '-cttaaattttCtTgaAt-3 ') ; MCP1 -L AN-5 ' (5,-CtTaAattttcttgaat-3,) ; and MCPl-LAN-even (5'-ctTaaatTttctTgaat-3'). All primers substituted with locked nucleic acids have an elevated melting temperature (Tm), while MCP1-LNA-5' primers show a particularly improved sequence accuracy (Phred Q30 counts). Thus, in other embodiments, the sequencing primer may comprise at least one locked nucleotide in its 5' end region, i.e., one-half, one-third or one-quarter of the 5' end of the 'sequencing primer. Prior to being provided to an optical detection element, the sequencing primer can be hybridized to a single sample nucleic acid (i.e., a nucleic acid to be detected comprising at least one end-linking primer). The sequencing primer can be hybridized to the sample nucleic acid by mixing the sample nucleic acid with a molar excess of the sequencing primer in a saline solution, such as 5xSSC (or 5xSSPE), 0.1% Tween20 ( Or 0.1% SDS) with 0.1% BSA buffer solution. The mixture can be heated to 65 ° C for at least 5 minutes and slowly cooled to room temperature to allow the primer/template to bond. Residual primers can be removed by suitable methods, such as molecular sieves. S{S 1 can be designed by suitable methods, including visual review of sequences or computer-assisted primer design, including end-linking and Both are introduced. Many software kits are available to assist with primer design, including DNAStarTM (DNAstar, Inc., Madison, WI), OLIGO 4.0 (National Biosciences, Inc.), Vectoe NTI® (Invitrogen), Primer Premier 5 (Premierbiosoft), and Prime3 ( Whitehead Institute for Biomedical Research, Cambridge, MA). Primer design, taking into account, for example, the molecularity to be sequenced, specificity, length, required melting temperature, 22 1378237 \ secondary structure, primer dimer, GC content, pH and ionic strength of the buffer solution and used Enzyme (ie, polymerase or ligase). See, for example, k〇 Joseph Sambrook and David Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd edition (2001). 2.1.3 Binding to the surface of the array After binding the sequencing primer to the nucleic acid to be sequenced, including one or more end-linking primers, the complex is prepared in a suitable buffer solution and supplied to the substrate. - Position the surface of the array and allow it to bond. In some embodiments, the sample nucleic acid (the nucleic acid to be detected and one or more end-linking primers) is attached to the conjugation site, and then sequencing or detection primers are provided. In other embodiments, the composite is first hybridized prior to being provided to a component. Only one binding site of a nucleic acid sample is known to be a valid position. In an embodiment, the complex is provided to the optical detection element and the sample nucleic acid is attached to a random linkage at the position array. In other embodiments, the sample nucleic acid can be provided to a predetermined junction on the array of locations by a suitable 'method, such as by a machine or liquid handling system. Suitable methods for attaching nucleic acids to a solid support are well known in the art. In some embodiments, the sample nucleic acid can be attached directly to a linker by covalent attachment, such as disulfide, thioester, guanamine, phosphonium phosphate or ester linkage; or by non-covalent linkage, for example Antibody/antigen, or biotin/biotin protein binding. In some embodiments, the sample nucleic acid can be attached to a conjugation site by an intermediate molecule. In some embodiments, the intermediate molecule of W can be a polymerase, such as a DNA polymerase. [S 1 23 1378237] As an illustrative example of direct, covalent nucleic acid attachment, Adeesietal (Nucleic Acid Research, 28:87 (2000)) modifies the 5' end of an primer to include an SH g group. According to the method of Adeesi et al., a nucleic acid can be prepared in 50 μM phosphate buffered saline (PBS) (NaPi: 0.1 M NaH2P04 pH 6.5, 0.1 M NaCl). The primer solution can then be supplied to a salted glass slide and cultured in a humidity control box at room temperature of about 5 % to allow the primer to bind to the wafer surface. After the binding reaction is completed, shake the pBS solution twice at room temperature for 5 minutes each time to remove unbound DNA. After 'month/leather, add 1 mM β-thiol to one. The PBS solution was used to rinse the position array at room temperature to deactivate the thiol groups of the unbound DNA. Again, the array surface is cleaned, for example once with 5xSSC, 0.1% Tween and once with 5xSSC buffer solution. Thus, in some embodiments, the method used by Adeesi et al can be used in the methods provided herein to attach a sample nucleic acid complex, for example, via a sequence primer or a 5' end of a sample nucleic acid to a linker. . In an alternate embodiment, the sample nucleic acid can comprise, for example, a biotinylated nucleotide' and can bind to an avidin on the surface of the linker. In another embodiment, the sample nucleic acid can comprise an antigenic moiety, such as BrdU or digoxigenin, which is bound to the binding site by an antibody (or a fragment thereof). By "antibody," it will be appreciated that this phrase includes fragments of immunoglobulin molecules, including, for example, one or more CDR regions; or variable heavy or variable light fragments. Antibodies may occur naturally, Recombinant or synthetic. Antibodies may also include, for example, multiple strains (P〇lyCl〇ne) and monoclone variants. In some embodiments, the antibodies are at least 1〇6, 1〇7, 8 9 10, 10 Μ. Or higher binding constant (ass〇ciati〇n constant) binds to 24 1378237 • its antigen. The structure, function and production of antibodies are well known in the art. See, for example, Gary Howard and Matthew Kasser,

Using Antibodies: A Practical Handbook CRC Press; 1st edition (2006)。 在又另一實施例中’藉由一聚合酶’例如DN A聚合 _，可將樣本核酸附著至連結位。熟悉此技藝人士可理解， 為了保留酵素功能，可得之資訊，例如酵素之一級、二級 與三級結構應被考慮。例如，Taq與Phi29聚合酶之結構為 • 本技術領域所知，分別參見：Kim et al., 376:612-616(1995)與 Kamtekar et al.，Μ〇/· Ce//， 16:609-618(2004)。將一聚合酶固定至一表面，而保持活性 之方法為本技術領域所知，且敘述於，例如美國專利公開 號 2〇〇8/〇199932,公開於 2008 年8月 21 日與 K〇rlachetal WAS 1G5:1176-1181(2GG8)。第8圖為—實施例之圖解表 示，其中樣本核酸（即，要被定序之核酸32、末端連結引 子34與疋序引子346)藉由一聚合酶38結合至一連結位 220，且聚合酶38係已結合至連結位22〇，藉由方法384， - 例如直接非共價吸附、一抗體、生物素或化學連結，例如 醯胺鍵。 在些貝把例中，一連結位之經乙搭（aidehyde)修飾之 表面係以含乙醛之矽烷(Silane)試劑處理。乙醛很快地與在 蛋白質上之一級胺反應以形成一 Sehiff，s鹼基連結。由於 除了 一般於ΝΑ端之更加反應性的α_胺外，很多蛋白質顯 露離胺酸於其表面上，所以其可以各種之方向附著至載 片，以允許蛋白質之不同側與於溶液中之其他蛋白質或小。 L ^ 1 25 1378237 分子反應。在另一實施例中，藉由uv光活化 (photoactivation)，一光 NHS(photoNHS) (— N-經基琥 ί白醯 亞胺缓酸鹽分子（N-hydroxy succimido carboxylate)以一碳 鏈連結器（linker)，連結至一疊氮硝基苯 (azidonitrobenzene))附著至一於元件上之經胺修飾之表 面。在這些實施例中，藉由消除氮，UV光激起一疊氮硝基 苯部分以產生高反應性氮烯(nitrene)。氮烯立即與元件表面 上之NH2反應以形成一聯氨（hydrazine)鍵。連結器之另一 端為NHS羧酸鹽，其與聚合酶表面上之離胺酸反應以產生 一醯胺共價鍵。在另一實施例中，於緩衝環境下，NHS羧 酸鹽部分與元件表面上之一級胺反應。UV光用來活化一疊 氮硝基苯部分且形成一高反應性氮烯為一電子不足基團 (electron deficient group)且立即與聚合酶表面上之離胺酸 殘基的一級胺反應。這些方法更詳細敘述於以下之實施例 4中。Using Antibodies: A Practical Handbook CRC Press; 1st edition (2006). In yet another embodiment, the sample nucleic acid can be attached to the conjugation site by polymerization of a polymerase such as DN A. Those skilled in the art will appreciate that in order to preserve the function of the enzyme, information such as one, two and three levels of enzymes should be considered. For example, the structure of Taq and Phi29 polymerase is known in the art, see: Kim et al., 376:612-616 (1995) and Kamtekar et al., Μ〇/· Ce//, 16:609 -618 (2004). A method of immobilizing a polymerase to a surface while maintaining activity is known in the art and is described, for example, in U.S. Patent Publication No. 2,8/199932, filed on Aug. 21, 2008, and K 〇rlachetal WAS 1G5: 1176-1181 (2GG8). Figure 8 is a graphical representation of an embodiment in which sample nucleic acids (i.e., nucleic acid 32 to be sequenced, end-linking primer 34, and leader primer 346) are bound by a polymerase 38 to a linker 220 and polymerized. The enzyme 38 has been bound to the linker 22 by method 384, such as direct non-covalent adsorption, an antibody, biotin or chemical linkage, such as a guanamine linkage. In some examples, the aidehyde-modified surface of a linker is treated with an acetaldehyde-containing silane (Silane) reagent. Acetaldehyde quickly reacts with a primary amine on the protein to form a Sehiff,s base linkage. Since many proteins exhibit lytic acid on their surface in addition to the more reactive alpha-amines generally at the terminal end, they can be attached to the slide in various directions to allow the different sides of the protein to be in the solution. Protein or small. L ^ 1 25 1378237 Molecular reaction. In another embodiment, by photoactivation of a uv light, a photo NHS (photoNHS) (N-hydroxy succimido carboxylate is linked by a carbon chain) A linker, attached to azidonitrobenzene, is attached to an amine-modified surface on the element. In these examples, by eliminating nitrogen, the UV light excites the monoazide nitrobenzene moiety to produce a highly reactive nitrene. The nitrene immediately reacts with NH2 on the surface of the element to form a hydrazine bond. The other end of the linker is an NHS carboxylate which reacts with the amine acid on the surface of the polymerase to produce a guanamine covalent bond. In another embodiment, the NHS carboxylate moiety reacts with a primary amine on the surface of the component in a buffered environment. The UV light is used to activate the monoazide nitrobenzene moiety and form a highly reactive nitrone which is an electron deficient group and reacts immediately with the primary amine of the amino acid residue on the surface of the polymerase. These methods are described in more detail in Example 4 below.

2.2定序形式 實施例所提供之生物分析系統可用來偵測與定序核 酸，藉由本技術領域已知的方法，如回顧於，例如美國專 利號 6, 946,249 與 Shendure et al·，#扣·〜ν· Gend. 5:335-44(2004)。在一些實施例中，定序方法依靠不是DNA 聚合酶就是DNA連接酶的專一性，且包括，例如鹼基延伸 定序（base extension sequencing)(單一驗基逐步延伸（single base stepwise extension))、藉由合成之多驗基定序（包括， 例如以經末端標誌之核苷酸來定序）與擺動定序（wobble 26 1378237 sequencing)，其為根據連結。所有方法通常需要—單股樣 本核酸，包括至少一要被附著至一連結位（不是直接就是 間接）之末端連結引子。之後定序開始實施於一定序引子 (連接麵疋序一般指錯定引子（anch〇r primer)，其對定序引 子提供類似物用途）。 對於所有定序形式而言，實施例提供可再定序之單一 分子。例如，於定序讀取完成後，可將定序引子與經延伸 之核苷酸自樣本核酸刪除、清洗元件並重複定序。在各種 實施例中，藉由相同或不同方法可重新執行再定序。藉由 將相同分子再定序，預期定序錯誤會下降成定序讀取次數 之次方。例如，若單次讀取每個鹼基之錯誤為1〇·3，之後 在兩次讀取後’其下降成（1(Γ3)2, ，1().6。此為單一分子 定序之特別的優點，由於用以定序之經修飾的核芽酸可失 去其“ δ；!'或阻礙基團產生，例如假的刪除。 2.2.1鹼基延伸定序：逐步延伸 在一些實施例中，實施例所提供之光偵測裝置可用來 執行鹼基延伸定序，如，例如美國專利號5,3〇2,5〇9中所揭 露。在-些實施例中’藉由將包括一要被定序之單股核酸 32、與要被疋序之單股核酸32之3,端結合之末端連結引 子34與黏合至其之一定序引子346的一部份雙重複樣本核 酸附著至一連結位220來開始驗基延伸定序’如第6圖中 所不。在-些實施例中’聚合酶38與經修飾之核苦酸之後 破提供至光偵測元件於適合之緩衝溶液卜在—些實施例 中，藉由在連結位之聚合酶，將縣減複合物附著 至連 t S 1 27 1378237 結位。在一些實施例中，核苷酸包括一共價連結之可偵測 標誌，例如一螢光標誌，與一阻礙基團以避免任何二次延 伸。因此，在一單一核苷酸加入至定序引子346之3’端之 後，定序中斷。 第7圖為一鹼基延伸反應之一實施例的第一步驟的圖 解顯示。將具有螢光阻礙基團364之核苷酸362藉由DNA 聚合酶38加至定序引子346之3’端。在一些實施例中，螢 光標誌扮演阻礙基團。在其他實施例中，它們為分開之部 分。一單一核苷酸合併於定序引子346之3’端，且藉由其 之標誌以對應之光偵測器210來鑑定。之後移除螢光標誌 與阻礙基團，例如，藉由化學或酵素分解，以允許鹼基延 伸之額外循環。在實施例中，可同時或連續地與在任何順 序中移除標誌、與阻礙基團。藉由編輯驗基加入順序，於3’ 至5’方向，一次一個鹼基推論出樣本核酸之序列。第9圖 顯示平行地將一些樣本核酸延伸、偵測與去阻礙/去標誌的 一循環。 一般而言，在逐步延伸時有兩個方式來辨認所加入之 核苷酸。於一第一例子中，四種核苷酸具有相同之可偵測 標誌，但以一預定之順序一次加入一個。經延伸之核苷酸 的鑑定藉由於延伸反應中所加入之核苷酸的順序來確認。 在於延伸時辨認經整合之鹼基的第二模式中，四種不同之 核苷酸同時加入，且各與一有區別可偵測之標誌結合。在 不同實施例中，標誌之激發或散發光譜及/或強度可不同。 藉由可偵測標誌之強度及/或波長（即，激發或散發光譜） 來確認於延伸中加入之核苷酸強度。這兩個方法論之實施 28 1378237 例呈現於實施例5中。 2.2.2藉由合成之定序：多步踢延伸（multi-step extension)2.2 Sequencing Forms The bioanalytical system provided by the examples can be used to detect and sequence nucleic acids by methods known in the art, as reviewed, for example, in U.S. Patent No. 6,946,249 and Shendure et al., #扣· ~ν· Gend. 5:335-44 (2004). In some embodiments, the sequencing method relies on the specificity of not being a DNA polymerase or DNA ligase, and includes, for example, base extension sequencing (single base stepwise extension), By synthetic multi-sequence sequencing (including, for example, sequencing with terminally labeled nucleotides) and wobble 26 1378237 sequencing, which is a pertinent linkage. All methods typically require a single strand of sample nucleic acid comprising at least one end-linking primer to be attached to a linker (not directly or indirectly). The sequencing then begins with a certain sequence of primers (the junction sequence is generally referred to as an anch〇r primer, which provides for analog use for sequencing primers). For all sequencing forms, the examples provide a single molecule that can be reordered. For example, after the sequencing reads are completed, the sequencing primers and the extended nucleotides can be deleted from the sample nucleic acid, the components washed, and the sequence repeated. In various embodiments, reordering can be re-executed by the same or different methods. By reordering the same molecules, it is expected that the sequencing error will fall to the power of the number of sequential reads. For example, if the error of reading each base in a single time is 1〇·3, then after two readings, it drops to (1(Γ3)2, ,1().6. This is a single molecule sequencing. A particular advantage, since the modified geranic acid used for sequencing can lose its "δ;!" or hinder the generation of groups, such as false deletions. 2.2.1 Base Extension Sequence: Progressively in some implementations In an example, the photodetecting device provided by the embodiments can be used to perform base extension sequencing, as disclosed in, for example, U.S. Patent No. 5,3,2,5,9. In some embodiments, Included is a single-stranded nucleic acid 32 to be sequenced, a terminally linked primer 34 to be ligated to the single-stranded nucleic acid 32 to be sequenced, and a partial double-replicating nucleic acid attached to a certain sequence of primers 346 bonded thereto. Up to a link 220 to initiate the base extension sequence as shown in Figure 6. In some embodiments, 'polymerase 38 and modified nucleotide acid are then provided to the photodetecting element for appropriate buffering. Solution In some embodiments, the county subtractive complex is attached to the t S 1 27 1378237 junction by a polymerase at the conjugation site. In some embodiments, the nucleotide comprises a covalently linked detectable marker, such as a fluorescent marker, and a barrier group to avoid any secondary extension. Thus, a single nucleotide is added to the sequencing primer 346. After the 3' end, the sequencing is interrupted. Figure 7 is a graphical representation of the first step of an embodiment of a one-base extension reaction. The nucleotide 362 having the fluorescent blocking group 364 is passed by DNA polymerase 38. Add to the 3' end of the sequencing primer 346. In some embodiments, the fluorescent labels act as blocking groups. In other embodiments, they are separate portions. A single nucleotide is incorporated into the sequencing primer 346-3 'End, and identified by its corresponding photodetector 210. The fluorescent marker and the blocking group are then removed, for example, by chemical or enzymatic decomposition to allow for additional cycles of base extension. In embodiments, the marker and the blocking group can be removed simultaneously or sequentially and in any order. The sequence of the sample nucleic acid is deduced one column at a time in the 3' to 5' direction by editing the sequence of addition. Figure 9 shows some samples in parallel A cycle of nucleic acid extension, detection, and deblocking/de-marking. In general, there are two ways to recognize the added nucleotide during the stepwise extension. In a first example, the four nucleotides have the same identity. The detectable marker is added one at a time in a predetermined order. The identification of the extended nucleotide is confirmed by the order of the nucleotides added in the extension reaction. The integrated base is recognized upon extension. In the second mode, four different nucleotides are added simultaneously and each is combined with a distinguishable detectable marker. In various embodiments, the excitation or emission spectrum and/or intensity of the marker may be different. The intensity and/or wavelength of the marker (i.e., excitation or emission spectrum) is detected to confirm the strength of the nucleotide added to the extension. The implementation of these two methodologies is shown in Example 5. 2.2.2 Sequence by synthesis: multi-step extension

在一些實施例中，藉由合成之定序可藉由多重不中斷 延伸（multiple uninterrupted extension)，例如沒有使用阻礙 基團來執行。在這些實施例中，藉由偵測在核三磷酸 (nucleoside triphosphates)水解後之焦構酸（pyrophosphate) 釋放，即，β與γ磷酸鹽複合物之釋放，來監測聚合反應。 可直接偵測複合物，例如藉由在複合物上之螢光部分，或 間接偵測複合物，藉由將焦磷酸與一化學或生物冷光偵測 系統結合。 在一些實施例中，藉由使用經末端-磷酸鹽標誌之核苷 酸（terminal-phosphate-labeled nucleotide)將核酸樣本定 序。經末端-磷酸鹽標誌之核苷酸與其使用方法之實施例序 述於，例如美國專利號7,361，466與美國專利公開號 2007/0141598，公開於2007年6月21日。簡單地說，將 核苷酸提供至實施例所提供之裝置’當於聚合中水解時， 藉由對應之光偵測器偵測經標誌之磷酸鹽。在一些實施例 中，所有之四種核苦酸包括有區別之標誌' 且可被同時加 入。在一些實施例令，核苷酸包括無法區別，即，相同之 標誌，且被以預定之順序連續地加入。連續、循環的加入 具有無法區別之標誌之核苷酸，依然允許多重、不中斷聚 合步驟，例如於均聚體序列中。In some embodiments, the sequencing by synthesis can be performed by multiple uninterrupted extensions, e.g., without the use of blocking groups. In these examples, the polymerization was monitored by detecting the release of pyrophosphate after hydrolysis of the nucleoside triphosphates, i.e., the release of the beta and gamma phosphate complexes. The complex can be detected directly, for example by combining the fluorescent moiety on the complex, or indirectly detecting the complex, by combining pyrophosphate with a chemical or biological luminescence detection system. In some embodiments, the nucleic acid sample is sequenced by using a terminal-phosphate-labeled nucleotide. Examples of end-phosphate-labeled nucleotides and methods of use thereof are described in, for example, U.S. Patent No. 7,361,466 and U.S. Patent Publication No. 2007/0141598, issued Jun. 21, 2007. Briefly, the nucleotides are provided to the device provided in the Examples. When hydrolyzed in the polymerization, the labeled phosphate is detected by a corresponding photodetector. In some embodiments, all four of the nucleotides include a distinctive signature' and can be added simultaneously. In some embodiments, the nucleotides include indistinguishable, i.e., the same, and are continuously added in a predetermined order. Continuous, cyclic addition of nucleotides with indistinguishable markers still allows multiple, uninterrupted polymerization steps, such as in homopolymer sequences.

29 1378237 2·2·3 連接酶定序(Ligase-Based Sequencing)29 1378237 2·2·3 Ligase-Based Sequencing

在其他實施例中，藉由連接酶定序，將一樣本核酸於 實施例所提供之光學偵測裝置上進行定序。連接酶定序方 法敘述於，例如美國專利號5,750,341、PCT公開 WOIn other embodiments, the same nucleic acid is sequenced on an optical detection device provided by the examples by ligase sequencing. The ligase sequencing method is described, for example, in U.S. Patent No. 5,750,341, PCT Publication WO

06/073504 與 Shendure et al. iSc/ewce，309:1728-1732 (2005) 中。在Shendure et al.的方法中，例如，一未知單股DNA 樣本可位於兩個末端連結引子的側面，且固定於一固體支 持物上。於未知序列之一特定位置（即，接近於一特定末 端連結引子之nth鹼基），可藉由將一所謂錨定引子（其為 〜定序引子之類似物）與末端連結引子黏合，且之後將4 個退化之九聚體的聯合提供至混合物來檢視。四個九聚體 白、有有區別之營光標遠、，且除了質問位置（query p0siti〇n) 其在所有位置皆為退化的，在質問位置各九聚體以一可區 別之鹼基一A、C、G或T來質問。將樣本清洗、螢光掃瞄 且鑑定質問之鹼基。之後自樣本核酸去除錨定引子與經連 、、、°之九聚體、清洗元件且重複步驟，以質問一不同位置。 有利地，此方法為非漸次的，即，驗基不需依次被質問。 因此，錯誤不會累計。此外，此方法可自不是5，就是3,方 向來質問核苷酸，即，不需標準之5,〜3，Ε)ΝΑ合成。藉由 此方法，可通常將總共約13個鹼基之一樣本核酸進行定 序。 2·3應用06/073504 and Shendure et al. iSc/ewce, 309: 1728-1732 (2005). In the method of Shendure et al., for example, an unknown single-strand DNA sample can be located on the side of the two end-linking primers and immobilized on a solid support. At a specific position in an unknown sequence (ie, an nth base close to a specific end-linking primer), a so-called anchoring primer (which is an analog of the ~-sequence primer) can be bonded to the end-linking primer, and The combination of the four degraded 9-mers is then provided to the mixture for review. The four nine-mers are white, and the difference is that the cursor is far away, and except for the question position (query p0siti〇n), it is degraded at all positions, and the 9-mer is a distinguishable base at the question position. Ask A, C, G or T. The sample is cleaned, scanned, and the base of the question is identified. Thereafter, the anchoring primer is removed from the sample nucleic acid, and the 9-mer, the cleaning element, and the steps are repeated to challenge a different position. Advantageously, the method is non-progressive, i.e., the test base need not be challenged in turn. Therefore, the error will not accumulate. In addition, this method can be used to question nucleotides from not 5, that is, 3, that is, without standard 5, ~3, Ε) ΝΑ synthesis. By this method, a sample nucleic acid of about 13 bases in total can be usually sequenced. 2·3 application

IS 生物分析系統可同時偵測數百萬之核酸片段。若各片 羊又為，例如1 〇〇〇個驗基長，一單一元件一次可獲得超過數 1378237 十億個驗基序列。此處所提供之元件的額外應用與方法將 於以下討論。 2.3.1整個基因體定序 生物分析系統可用來執行，例如病毒、細菌、真菌、 真核生物或脊椎動物，例如哺乳動物，例如人類，之整個 或部分基因體定序。 為了定序’可將基因體DNA切割成至少2〇、5〇、1 〇〇、 • 200、300、500、800、1200、1500個核苷酸或更長之片段。 在一些實施例中’經切割之基因體DNA可為自2〇至50、 自 20 至 100、自 20 至 500、自 20 至 1〇〇〇、自 500 至 12〇〇 或自500至1500個核苦酸長。在一些實施例中，為了如上 述之定序，具有經結合之末端連結引子之要被定序的核 酸’被製成單股、與定序引子黏合且被提供至實施例所提 供之元件。 2.3.2 基因表現研究(Gene Expression Profiling) ' 在其他貝細*例中’為了基因表現研究，生物分析系統 可用來將cDN A定序。例如’藉由測量於元件上被彳貞測之 特定序列的相關頻率’可將mRNA程度進行定量。可將數 百萬cDNA分子平行定序於實施例所提供之一元件上。若 一細胞平均包含350,〇〇〇個mRNA分子，於一百萬個定序 反應中，預期將存在於正好每個細胞一複製的一轉錄物 (transcript)定序三次。因此，實施例所提供之元件適合具有 單一數目靈敏度之單一分子定序。The IS Bioanalytical System detects millions of nucleic acid fragments simultaneously. If each piece of sheep is again, for example, 1 验 base length, a single component can obtain more than 1,378,237 billion base sequences at a time. Additional applications and methods for the components provided herein will be discussed below. 2.3.1 Whole Genome Sequencing Bioanalytical systems can be used to perform whole or partial genotyping of viruses, bacteria, fungi, eukaryotes or vertebrates, such as mammals, such as humans. For sequencing, the genomic DNA can be cleaved into fragments of at least 2, 5, 1, 2, 200, 300, 500, 800, 1200, 1500 nucleotides or longer. In some embodiments, the 'cutted genomic DNA can be from 2 to 50, from 20 to 100, from 20 to 500, from 20 to 1 , from 500 to 12, or from 500 to 1500. The acid is long and bitter. In some embodiments, for sequencing as described above, the nucleic acid' to be sequenced with the bound end-linking primers is made into a single strand, bonded to a sequencing primer and provided to the elements provided by the examples. 2.3.2 Gene Expression Profiling 'In other shells' cases] For gene expression studies, bioanalytical systems can be used to sequence cDN A. For example, the degree of mRNA can be quantified by measuring the relative frequency of a particular sequence that is speculated on the element. Millions of cDNA molecules can be sequenced in parallel to one of the elements provided in the examples. If a cell contains an average of 350, mRNA mRNA molecules, in one million sequencing reactions, it is expected that a transcript that replicates exactly one cell will be sequenced three times. Thus, the elements provided by the examples are suitable for single molecule sequencing with a single number of sensitivities.

t SI 31 1378237t SI 31 1378237

cDNA合成為本技術領域所熟知，且一般包括總RNA 萃取與視需要而定之mRNA豐富。藉由步驟，包括，例如： 為了第一股合成之反轉錄；RNAse處理以移除殘餘RNA ; 第一股之隨機六聚體起始(priming)與藉由DNA聚合酶之 第二股合成，來由mRNA產生cDNA。合成之cDNA適合 在實施例所提供之元件上進行定序。分離與製備DNA與 RNA兩者之方法為本技術領域所熟知。參見，joseph Sambrook and David Russell, Molecular Cloning: AcDNA synthesis is well known in the art and generally includes total RNA extraction and mRNA enrichment as desired. By steps including, for example: reverse transcription for the first synthesis; RNAse treatment to remove residual RNA; first random hexamer priming and second synthesis by DNA polymerase, To generate cDNA from mRNA. The synthesized cDNA is suitable for sequencing on the elements provided in the examples. Methods for isolating and preparing both DNA and RNA are well known in the art. See, joseph Sambrook and David Russell, Molecular Cloning: A

Cold Spring Harbor Laboratory Press, 3rd edition (2001)。 在一些實施例令，藉由於美國專利號6,812,005與 7,361,488中所述方法可將cDNA進行定序。簡短來說， cDNA與接合物(adapter)聚核酸連結’以專一限制酵素來處 理接合物’且最後經處理之核酸與附著於實施例所提供元 件之連結位的互補寡核苷酸結合。在實施例中，接合物分 子為末端連結引子。 在一些實施例中’ mRNA之多腺嘌呤尾巴(p〇ly a tail) 可做為一適合之末端連結引子，其與一多胸腺嘧啶(p〇ly τ tail)定序引子互補。 2.3.3偵測及/或測量結合之相互作用 在其他實施例中’生物分析系統可用來偵測各種結合 相互作用，包括，例如DNA/DNA、RNA/RNA或DNA/RNA 鹼基配對、核酸/蛋白質相互作用、抗原/抗體、受器/配體 結合與酵素/受質結合。一般而言，一樣本分子附著於包括 32 1378237 - 一鑑定核酸標籤（identifying nucleic acid tag, ID)之連結分 子。在一些實施例中，連結分子更包括一與樣本分子結合 之捕獲分子。連結分子也包括用以與連結位結合之工具， 例如一促進共價化學連結之部份，例如，雙硫、硫酯、醯 胺、磷酸二酯或酯連結；或藉由非共價連結，例如抗體/抗 原或生物素/抗生物素蛋白結合。在一些實施例中，藉由ID 標籤將連結分子附著至陣列。 ' 將一樣本分子提供至一元件，且藉由其之連結分子將 Φ 其附著於一隨機連結位，例如藉由與一位於連結分子上之 捕獲分子結合。在一些實施例中，將樣本分子與連結分子 混合、允許其結合、且之後提供至實施例所提供之一元件。 在一些實施例中，連結分子先被提供至元件，允許其附著 至一連結位，而之後提供樣本分子。再來，藉由鑑定經結 合之樣本分子的方法來偵測ID(例如，藉由雜合或定序）。 複數個樣本分子種類可附著至相同之陣列，且藉由其之標 誌來區分，而使用與其結合之捕獲分子獨特的ID，可以其 B 之結合相互作用為特徵。因此，在一些實施例中，偵測一 - 經標誌之樣本分子的方法包括，藉由包括一核酸標籤(ID) 之連結分子，來連結一樣本分子至一元件之連結位、執行 ID之核酸定序與偵測經標誌之樣本分子的步驟。在實施例 中，核酸定序為鹼基延伸定序。在一些實施例中，核酸定 序係擇自連接酶定序或經末端-磷酸鹽標誌之核苷酸定序。 藉由使用核苷酸“小段(bits)”，上至4n個具區別之捕獲 分子可被附著於與鑑定於生物分析系統上，其中η為一自 然數字顯示被定序之ID的長度。例如，5個核苦酸可提供 [ 33 1378237 超過一千個獨特ID，而12個核苷酸提供超過一千六百萬 個組合。例如，連結分子附著至一元件而其位置藉由偵測 其對應之ID標籤來確認。之後連結分子作為探針以，例如 調查與一或多個經標誌之樣本分子的結合相互作用。即， 具有一或多個連結分子附著至其的一元件可作為一探針陣 列。 在實施例中，一經標誌之樣本分子為經螢光標誌、。當 與連結分子之捕獲分子結合時，藉由對應於被連結分子附 著之連結位的光偵測器，來偵測經標誌、之樣本分子。因此， 在一些實施例中，方法可更包括提供一經標誌之樣本分子 至一元件及偵測經標誌之樣本分子的步驟。在實施例中， 元件具有附著至連結位之連結分子，而連結分子包括一附 著至連結位之核酸標籤(ID)。可將多重標誌樣本分子同時 提供至一探針陣列且可藉由其標誌來區分，例如藉由其螢 光標誌之強度及/或波長。在一經標誌質問分子之所給予的 濃度下，基於動力學（例如，接合（docking)/與未接合 (undocking))與統計學（例如，在任何給予之時間，樣本 分子之部分為結合或為結合之狀態）兩者可干擾介於樣本 分子與經標誌質問分子之間的結合相互作用的分離常數。 在一些實施例中，一連結分子之ID為至少5、10、15、 20、25、30、40、50、75、90、100、150、200 或更多個 核苷酸長。在一些實施例中，ID為自5至10、20、40、80 或160 ;或自10至20或50 ;或自20至35個核苷酸長。 ID包含一獨特之核酸序列，即，一要被偵測之核酸。在實 施例中，獨特之核酸序列可為至少1、2、4、6、8、10、 34 1378237 12、14、16、20、24、30或更多個核苷酸長。在一些實施 例中，獨特之核酸序列為自4至1〇、12、15或20，或自 10至2〇個核苷酸長。ID包括至少一末端連結引子，即， ID包含一序列其與一定序引子互補，此序列在一些實施例 中，為經修飾的，以附著至一連結位’例如藉由包含一經 生物素化之核苷酸。在一些實施例中，ID之末端連結引子 部分為3’端至獨特核酸序列。在一些實施例中’其為5,端 至獨特核酸序列。在又另一實施例中’末端連結引子出現Cold Spring Harbor Laboratory Press, 3rd edition (2001). In some embodiments, the cDNA can be sequenced by the methods described in U.S. Patent Nos. 6,812,005 and 7,361,488. Briefly, the cDNA is conjugated to an adapter nucleic acid' to treat the conjugate by a specific restriction enzyme' and the final treated nucleic acid is conjugated to a complementary oligonucleotide attached to the junction of the elements provided in the Examples. In an embodiment, the conjugate molecule is an end-linking primer. In some embodiments, the mRNA polyp tail can be used as a suitable end-linking primer that is complementary to a poly-thymidine (p〇ly τ tail) sequencing primer. 2.3.3 Detection and/or Measurement of Binding Interactions In other embodiments, a bioanalytical system can be used to detect various binding interactions, including, for example, DNA/DNA, RNA/RNA or DNA/RNA base pairing, nucleic acids. /protein interactions, antigen/antibody, receptor/ligand binding combined with enzyme/substrate. In general, the same molecule is attached to a linker comprising 32 1378237 - an identifying nucleic acid tag (ID). In some embodiments, the linker molecule further comprises a capture molecule that binds to the sample molecule. Linking molecules also include means for binding to the linking sites, such as a moiety that promotes covalent chemical bonding, for example, a disulfide, thioester, guanamine, phosphodiester or ester linkage; or by non-covalent linkage, For example antibody/antigen or biotin/avidin binding. In some embodiments, the linker molecules are attached to the array by an ID tag. The same molecule is provided to a component, and the linker molecule thereof attaches Φ to a random link, for example by binding to a capture molecule located on the linker molecule. In some embodiments, the sample molecules are mixed with the linking molecules, allowed to bind, and then provided to one of the elements provided by the embodiments. In some embodiments, the linker molecule is first provided to the element, allowing it to attach to a linker, and then providing the sample molecule. Again, the ID is detected by identifying the combined sample molecules (e. g., by hybridization or sequencing). A plurality of sample molecular species can be attached to the same array and distinguished by their logo, and the unique interaction ID of the capture molecule combined with them can be characterized by the binding interaction of B. Thus, in some embodiments, the method of detecting a labeled sample molecule comprises linking the nucleic acid of the same molecule to a link of the element by a linker comprising a nucleic acid tag (ID) The step of sequencing and detecting the labeled sample molecules. In an embodiment, nucleic acid sequencing is base extension sequencing. In some embodiments, the nucleic acid sequence is selected from ligase sequencing or nucleotide sequencing via a terminal-phosphate label. By using nucleotide "bits", up to 4n distinct capture molecules can be attached to and identified on the bioanalytical system, where n is a natural number showing the length of the ID being sequenced. For example, 5 nucleotides can provide [33 1378237 more than a thousand unique IDs, while 12 nucleotides provide more than 16 million combinations. For example, a linker molecule is attached to a component and its position is confirmed by detecting its corresponding ID tag. The molecule is then linked as a probe to, for example, investigate the binding interaction with one or more labeled sample molecules. That is, an element having one or more linking molecules attached thereto can serve as a probe array. In an embodiment, the labeled sample molecule is a fluorescent marker. When combined with the capture molecule of the linker molecule, the labeled sample molecule is detected by a photodetector corresponding to the linker to which the linker is attached. Thus, in some embodiments, the method can further include the steps of providing a labeled sample molecule to a component and detecting the labeled sample molecule. In an embodiment, the element has a linker molecule attached to the linker and the linker molecule comprises a nucleic acid tag (ID) attached to the linker. Multiple-marker sample molecules can be provided simultaneously to a probe array and can be distinguished by their signature, such as by the intensity and/or wavelength of their fluorescent signature. At a concentration given by the marker molecule, based on kinetics (eg, docking/undocking) and statistics (eg, at any given time, portions of the sample molecule are bound or The state of binding) both can interfere with the separation constant between the binding interaction between the sample molecule and the labeled interrogating molecule. In some embodiments, the ID of a linker molecule is at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 90, 100, 150, 200 or more nucleotides in length. In some embodiments, the ID is from 5 to 10, 20, 40, 80 or 160; or from 10 to 20 or 50; or from 20 to 35 nucleotides in length. The ID contains a unique nucleic acid sequence, ie, a nucleic acid to be detected. In embodiments, the unique nucleic acid sequence can be at least 1, 2, 4, 6, 8, 10, 34 1378237 12, 14, 16, 20, 24, 30 or more nucleotides in length. In some embodiments, the unique nucleic acid sequence is from 4 to 1 , 12, 15 or 20, or from 10 to 2 nucleotides in length. The ID includes at least one end-linking primer, ie, the ID comprises a sequence that is complementary to a sequence of primers, which in some embodiments is modified to attach to a linker', for example by including a biotinylated Nucleotide. In some embodiments, the end joining primer portion of the ID is from the 3' end to a unique nucleic acid sequence. In some embodiments, it is 5, end to a unique nucleic acid sequence. In yet another embodiment, the 'end link primer appears

於獨特之核酸列的3,與5,端兩者。 在實施例中，樣本分子與捕獲分子包括係擇自—碳水 化合物、脂質、蛋白質、胜肽、抗原、核酸、荷爾蒙、小 α機刀子（例如藥學上的）或維他命部分或其組合之部分。 沒些部八、 如h 77可為自然發生（例如經生物化學純化）或合成（例 包$化學合成或重組產生）。此外，這些基質可沒有包含、 保^〜些或全部非天然成分（例如非天然胺基酸、阻礙或 白質基團等）。在實施例中’一樣本分子或捕獲分子為蛋 、’例如生長因子、胜肽抗原、抗體或受器。 域所t多結合核酸至連結分子或連結位的方法為本技術領 <33ι 〇 ’如回顧於’例如美國專利公開號2004/0038331中。 上公開揭露形成蛋白質募核苷酸結合物於一固態支持物 美國專利號4,74M 11提供結合蛋白質至一核酸之3， 貫施例。其中’末端轉移酶首先用來將核糖殘基加 之3’部分。一過蛾酸氧化（peri〇(jate oxidation)反 應之德a , 无屐生一 3’乙醛基團於一核酸上，3,乙醛基團之後與 蛋白暂+ 胺基團形成共價鍵結。當蛋白質結合至ID之3 ’ [S 1 35 1378237 端時，係經由ID之5’端附著至連結位。 在一些實施例中’ 一捕獲分子，例如一蛋白質，連結 至一 ID之5’端。在這些實施例中’id之3,端或一定序引 子之5’端係用來將捕獲分子附著至一連結位。美國專利號 6,013,434 ’例如揭露寡核苦酸_聚酿胺結合物，其中連接係 藉由养核皆酸之5’端。美國專利號6，197,513揭露PNA與 DN A兩者經由核酸之5 ’端結合至一具有缓酸部分之分子， 例如蛋白質。PNA與DNA分子包含芳香胺(aryiamine)或氮 氧乙醯(aminooxyacetyl)部分。美國專利號6，153,737揭露 包含至少一 2經g此化之核皆的券核苦酸，適合與多種分 子結合。 2.3.4額外之偵測方法In the unique nucleic acid column 3, and 5, both. In embodiments, the sample molecules and capture molecules include portions selected from the group consisting of - carbohydrates, lipids, proteins, peptides, antigens, nucleic acids, hormones, small knife (e.g., pharmaceutically) or a vitamin moiety, or a combination thereof. No part 8. For example, h 77 may be naturally occurring (for example, biochemically purified) or synthesized (for example, chemical synthesis or recombinant production). In addition, these matrices may contain no, some or all of the non-natural ingredients (e.g., unnatural amino acids, hindered or white matter groups, etc.). In the examples, the present molecule or capture molecule is an egg, such as a growth factor, a peptide antigen, an antibody or a receptor. A method in which a domain binds a nucleic acid to a linker molecule or a linker is in the art of <33>'' as reviewed in ', for example, U.S. Patent Publication No. 2004/0038331. The above disclosure discloses the formation of a protein-raised nucleotide conjugate in a solid support. U.S. Patent No. 4,74M11 provides a binding protein to a nucleic acid of 3, a specific example. Wherein the 'endotransferase is first used to add a ribose residue to the 3' portion. Oxidation of molybdenum (perate (jate oxidation) reaction a, no 3' acetaldehyde group on a nucleic acid, 3, acetaldehyde group and covalent bond with protein temporary + amine group When the protein binds to the 3' [S 1 35 1378237 end of the ID, it is attached to the junction via the 5' end of the ID. In some embodiments, a capture molecule, such as a protein, is linked to an ID 5 'End. In these examples, the ''3', the 5' end of the end or a certain sequence of primers is used to attach the capture molecule to a linker. U.S. Patent No. 6,013,434 ', for example, discloses oligo-picoic acid-polymerization The linker is made by the 5' end of the nutrient acid. U.S. Patent No. 6,197,513 discloses that both PNA and DN A bind to a molecule having a slow acid moiety, such as a protein, via a 5' end of the nucleic acid. PNA and The DNA molecule comprises an aryiamine or an aminooxyacetyl moiety. U.S. Patent No. 6,153,737 discloses a nucleus acid comprising at least one of the nucleus of the nucleus, which is suitable for binding to a plurality of molecules. 4 additional detection methods

2.3.4.1FRET 在一些實施例中，於一本發明所提供之光偵測裝置上 偵測刀子’藉由Forster共振能量轉移（F〇rster resonance energy transfer，FRET) ’有時也知為螢光共振能量轉移 (Fluorescence resonance energy transfer，FRET)。如本技術 領域所知，當一經激發之提供者分子非放射性轉移能量至 一接受者分子時，其散發能量，一般為光時，FRET發生。 FRET可幫助減低背景訊號，藉由，例如提供對於要偵測分 子之介於有效激發與散發波長之間的較大光譜間隔eFRET 吊用來偵測接近之分子相互作用，由於其功效衰敗隨著介 於提供者與接受者分子間之距離的第六能量。例如，Zhang 36 1378237 : et a1· (#如’施阶’Ά，4:826-31 (2005))藉由 FRET 偵測核 r 酸雜合。其中，一經生物素化之核酸標的與一覆蓋抗生物 素蛋白之量子點（quantum dot)提供者結合，其之後激發一 經Cy5結合之DNA探針。在本發明一些實施例中，一經 標誌之捕獲分子與經標誌之樣本分子可形成一用以藉由 FRET來偵測之提供者/接受者（或反之亦然） 配對。 在實施例所提供之核酸定序的一些實施例中，對於一 • 附著至一聚合酶或連接酶之提供者帶色團（chromophore)， • 勞光標誌核苷酸扮演-接受者帶色團。因此，於這些實施 例中’位於聚合酶或連接酶上之提供者帶色團激發一核酸 上之的接受者帶色團，而核酸要被聚合於或連接至樣本核 酸上。由於在FRET功效中之快速下降，不接近聚合酶之 核苦酸不被激發。在-些實施例中，提供者分子為，例如 另-螢光團，例如-量子點。量子點，例如，半導體量子 點為本技術領域所知且敘述於，例如國際公開號W0 _ 〇3/〇03015。結合量子點至，例如生物分子的方法為本技術 領所知如回顧於，例如Mednitz et al (伽咖她⑽油， 4·235 46 (2005))與美國專利公開號遍嶋㈣6盥 2〇_087843分別公開於2006年3月20日與2008年4月、 17日。在-些實施例中，量子點與—舰聚合酶分子結 合’其更進敘述於以下之實闕3。如先前所討論， 為了將，素與連！α位結合’熟悉此技藝人士可毫無疑問地 理解，當將螢光團結合至，例如一 DNa聚合酶或連接酶時 必需小心’以藉由減輕結合螢光團至酵素之-級、二級盘 三級結構上的任何影響來保持酵素功能。 [S3 37 1378237 2.3.4.2多光子激發 在一些實施例中，由兩個或更多之光子激發一帶色 團。例如’在一些實施例中，於FRET中，不是提供者就 是接受者帶色團之激發係經由兩個或更多之光子。兩個光 子與多光子激發更進一步敘述於，例如美國專利號 6,344,653 與 5,034,613 中。 2·3·4·3 時間解析 貞測(Time Resolved Detection) 在一些實施例中’可調整本發明所提供之光源與光偵 測器以具有一特徵相移(characteristic phase shift)。使用本 技術領域已知的方法，例如’如於美國專利公開號 2008/0037008公開於2008年2月14日，揭露了，散發自 於實施例所提供之元件上被偵測之分子的光，可藉由一對 應光彳貞測器來測量，而無來自激發光源的干擾。 3.使用生物分析系統之生物分子分析服務 實施例也提供一藉由使用根據本發明生物分析系^ f供生物分子分析服務的方法。在—些實施例中，方法 =自-服務請求者提供—包括—要被分析之生物分子的 八批供者’與服務請求者接受來自服務提供者 驟’其中藉由使用本發明所提供之元件產 二二:協考慮來執行方法， 服務請求者與㈣提供k間， is ] 38 1378237 • 運送。在一些貫施例中，服務提供者或賣主可地理性位於 r 美國之外的領土，例如於另一國家。 除非以其他方式指出，使用於說明書，包括申請專利 範圍中之成分、反應條件等的所有數字表現量，可被理解 . 為在所有情況下藉由措辭“約，，來修飾。 • 除非以其他方式指出，發出一系列要素之措辭‘‘至少’， 可被瞭解為意指於系列中之每一要素。 【實施例】 實施例1: 一高通量生物分析系統之建構 藉由參閱第1-4圖’製造一生物分析系統1之方法將 會於以下進行詳述。首先，藉由商業上可得之用於一般邏 輯與光學元件的0.25 μιη半導體製造製程一起提供一矽基 板10與形成於基板1〇上表面上之複數個光偵測器21〇。 光偵/貝]器210為光二極體光子偵測器，各具有一 24 μπι之 直與一 452 μιη2之露出面積。將各光偵測器排列成彼此 鄰接以便光偵測器210之512行與512列的陣列形成於基 板10上。 “複數個控制電路215形成於基板10之上表面之沒有形 成光偵測态210的位置上。於此實施例中，一控制電路215 對應於一光债測器210 ’以便控制其對應之光偵測器210 之操作及控制介於光偵測ϋ 210與賴記錄祕2間之通 1 39 1378237 在此實施例中，一濾層240形成於光偵測器210之上 表面與控制電路215上。在形成濾層240前，對光偵測器 210之上表面與控制電路215實施全面平坦化（global planarization)製程。濾層240包括複數個次層。在此實施 例中，遽層240可包括形成於一基板材料(base substrate material)上之大量次層，其可藉由形成（forming)、放置 (placing)或沈積一基板材料，例如Si02於基板1〇上來提 供。在一實施例中’可實施半導體製造或沈積方法。例如， 沈積方法可包括’但不限於，物理沈積、化學沈積、分子 束磊晶（molecular beam epitaxy，MBE)與原子層沈積（at〇mic layer deposition, ALD)。 在一些實施例中’基板材料可包括一或多個凹陷形成 於一表面上。在一些情況中，凹陷可為非平面，例如具有 一經定義之半徑的半球形或弧線形。例如，參見第3a圖， 於一實施例中’最接近基板10之濾層240之次層的半徑可 為約6.1 μιη ’且可自1 μπι變化至100 μιη。於一系統中之 凹陷的尺寸可根據系統之設計與應用及所對應之針孔的尺 寸來變化。在一實施例中，根據許多考量可變化半徑例如， 系統之應用、設計、特徵、光偵測器及/或光源與要被偵測 之生物分子的特徵。在一些實施例中，凹陷可為基板形成 製程之部分或藉由使用一般半導體製程移除技術來移除基 板材料的一部份來形成。例如，移除方法可包括溼蝕刻、 乾蝕刻或其方法。在其他實施例中，可在基板材料形成時， 使用壓力或揞壓來形成凹陷。 IS 3 一旦如上述形成濾層240之基板材料，系列之次層可 40 1378237 ' 形成於基板材料之頂部上以產生如第3a圖所示之濾層 ^ 240。在一些例子中，藉由沈積具有不同介電特性之材料的 替代層可形成次層。例如，藉由首先於基板材料上沈積一 具有較高折射率之次層於經平坦化之光偵測器21 〇之上表 面與控制電路215上可形成濾層240。之後，一具有較低 折射率之次層沈積於已形成之具有較高折射率之次層上。 在一些實施例申，具有較高折射率之次層可包含Nb205，. 而具有較低折射率之次層可具有Si〇2。藉由接連地沈積較 ^ 高折射率與較低折射率之次層，形成遽層240直到一大量 之次層已沈積於基板材料上。在此實施例中，濾層240包 括69個次層。基於各種考量，例如系統之應用、設計、特 徵、光偵測器及/或光源，可改變次層之數目、其折射率、 其材料及/或其他參數。 參見第5圖，其顯示一概括濾、層240之一實施例建構 的表格。在第5圖中，一較低編號之次層提供一較接近濾 層240之底部表面的次層，而一較高編號之次層提供一較 ® 接近濾層240之上表面的次層。第5圖中所示，於此實施 • 例中，濾層240之奇數次層由，例如五氧化二銳(Nb205)所 製成’其具有一較高之折射率。偶數次層由例如二氧化石夕 (Si〇2)所製成，其具有一較低之折射率。次層可藉由使用 一濺鍍系統來形成，濺鍍系統之例子包括Model No. RAS 110 of Radical Assisted Sputtering Series，由 Shincron Co” 1^(1.(8110^8&\¥&-1〇1，丁〇1^〇，认？八]^)所製造。於此實施例中 之各層之厚度也提供於第5圖之表格中。合成之濾層240 對於螢光團Cy5之螢光為高度透明，而對於散發自氦氖 [S] 41 1378237 (Helium-Neon)雷射之光在約633 nm波長為低穿透度，氦 氖雷射做為一外部光源以激發螢光團Cy5。 再次參見第2與4圖，具有針孔235之遮蔽薄板230 形成於濾層240上。在一實施例中，針孔235之尺寸可為 約0.2 μιη ’且可自〇 1 變化至1 pm。基於系統之設計 與應用及對應之據層的尺寸可改變於系統中之針孔尺寸。 在一實施例中，基於許多考量，例如系統之應用、設計、 特徵、光偵測器及/或光源與要被偵測之生物分子的特徵可 改變半徑。製造具有針孔235之遮蔽薄板230於濾層240 或基板10上的一製程將於以下詳述。 首先’藉由’例如旋塗一光阻材料於濾層240上，形 成一光阻層於濾層24〇上（若濾層24〇為視需要形成於光 偵測器210之上表面與控制電路215上）或於經平坦化之 光偵測裔210之上表面與控制電路215上（若不形成濾層 240)。之後，將光阻顯影以在針孔區形成光阻圖案。藉由 使用光罩覆蓋針孔區，且將光阻曝光以使只有由光罩覆蓋 之區域維持於濾層240或經平坦化之光偵測器21〇之上表 面與控制電路215上來形成光阻圖案。 之後’金屬層沈積於已形成光阻圖案之濾層24〇上。 在此實施例中，金屬層可包括鉻(Cr)，藉由執行一磁控濺 鍍製程（magnetron sputtering process)，其沈積於濾層 24〇 或經平坦化之光偵測器210之上表面與控制電路215上。 接著’移除於針孔區上之金屬層部分與於針孔區中之 光11且圖案’藉此形成具有針孔235之遮蔽薄板230。 或者’藉由首先沈積一金屬層（例如’ Cr )於濾層240 i j 1 42 1378237 • 上，之後形成一罩幕於金屬層上，藉此將金屬層之上表面 之部分曝光，來形成遮蔽薄板230。之後將金屬層曝光之 % 部分進行蝕刻，直到露出濾層240，藉此形成針孔於金屬 層上。之後，將罩幕自金屬層移除且形成具有針孔235之 遮蔽薄板230於濾層240上。在一些實施例中，以Si02填 滿由介於遮蔽薄板230與最接近遮蔽薄板230之次層之間 的空間所形成之半球，且在形成遮蔽薄板230前，將半球 磨光以平坦表面。而上述金屬層具有形成於濾層240之半 Φ 球中央上的針孔235。 再次參見第2與4圖，於此實施例中，藉由在針孔235 或凹處450填入一支持材料來形成連結位220。支持材料 可為對於散發自螢光團36之螢光為透明之聚合物或無機 材料。 再次參見第1圖，雖然只顯示12個光學偵測裝置20。 可以瞭解的是，至少一萬個光學偵測裝置20可形成於基板 10上。例如，於此實施例中，各光學偵測裝置20具有一 I 半徑為約5 μιη或更小之圓形形狀，其可佔據約100 μιη2之 面積。對於具有1平方英叶（即，2.54 cm乘以2.54 cm) 之面積的基板10而言，建構大於六百萬個光學偵測裝置 20於基板10上為可能的。藉由同時操作那些六百萬個光 學偵測裝置20，可以高通量來偵測生物分子。 實施例2 :以高通量生物分析系統之生物分子的附著 與偵測 [S ] 43 13782372.3.4.1 FRET In some embodiments, detecting a knife 'F〇rster resonance energy transfer (FRET)' is sometimes also known as fluorescence on a photodetecting device provided by the present invention. Fluorescence resonance energy transfer (FRET). As is known in the art, FRET occurs when an excited donor molecule non-radioactively transfers energy to a recipient molecule, which emits energy, typically light. FRET can help reduce the background signal by, for example, providing a large spectral separation between the effective excitation and emission wavelengths of the molecule to be detected. The eFRET sag is used to detect the proximity of the molecular interaction due to its efficacy decay. The sixth energy between the distance between the provider and the recipient. For example, Zhang 36 1378237: et a1· (#如', 施, 4:826-31 (2005)) detects nuclear heterozygous by FRET. Wherein, a biotinylated nucleic acid target is combined with a quantum dot provider covering the avidin, which is followed by a Cy5-bound DNA probe. In some embodiments of the invention, a labeled capture molecule and a labeled sample molecule can form a pair of providers/recipients (or vice versa) for detection by FRET. In some embodiments of the nucleic acid sequencing provided by the embodiments, for a chromophore attached to a polymerase or ligase provider, • Lloyd's logo nucleotide-receptor with chromophore . Thus, in these embodiments, a provider located on a polymerase or ligase chromophore excites a recipient with a chromophore, and the nucleic acid is polymerized or attached to the sample nucleic acid. Due to the rapid decline in FRET efficacy, the nucleotide acid that is not close to the polymerase is not excited. In some embodiments, the donor molecule is, for example, an additional-fluorescent group, such as a quantum dot. Quantum dots, for example, semiconductor quantum dots are known in the art and are described, for example, in International Publication No. WO _ 〇 3/〇03015. Methods for binding quantum dots to, for example, biomolecules are known to the art as reviewed, for example, Mednitz et al (Gaga Her (10) Oil, 4·235 46 (2005)) and U.S. Patent Publication No. 4 (6) 6盥2〇 _087843 was published on March 20, 2006 and April, April 17, respectively. In some embodiments, the quantum dots are combined with a -carrier polymerase molecule, which is further described in the following. As discussed earlier, in order to be, Su and even! Alpha-Bound Combinations A person skilled in the art will undoubtedly understand that when fluorophores are bound to, for example, a DNa polymerase or ligase, care must be taken to reduce the binding of the fluorophore to the enzyme-level, Any effect on the tertiary structure of the plate to maintain the enzyme function. [S3 37 1378237 2.3.4.2 Multiphoton Excitation In some embodiments, a band of chromons is excited by two or more photons. For example, in some embodiments, in FRET, it is not the provider that the recipient's chromophore excitation system passes two or more photons. Two photons and multiphoton excitation are further described, for example, in U.S. Patent Nos. 6,344,653 and 5,034,613. 2·3·4·3 Time Resolved Detection In some embodiments, the light source and photodetector provided by the present invention can be adjusted to have a characteristic phase shift. The use of methods known in the art, for example, as disclosed in U.S. Patent Publication No. 2008/0037008, issued Feb. 14, 2008, discloses the disclosure of the light of the detected molecules on the elements provided in the examples, It can be measured by a corresponding optical detector without interference from the excitation source. 3. Biomolecular Analysis Service Using a Bioanalytical System The embodiment also provides a method for providing biomolecule analysis services by using the bioanalytical system according to the present invention. In some embodiments, the method = provided by the service requester - includes - eight batches of donors of the biomolecule to be analyzed 'and the service requester accepts from the service provider" wherein the invention is provided by using the invention Component production 22: Co-consider to implement the method, service requester and (4) provide k, is ] 38 1378237 • Shipping. In some embodiments, the service provider or vendor may be geographically located outside of the United States, such as in another country. Unless otherwise stated, all numerical expressions used in the specification, including the ingredients, reaction conditions, etc. in the scope of the patent application, are to be understood. In all cases, by the wording "about," are modified. The method states that the wording 'at least' of a series of elements can be understood to mean each element of the series. [Embodiment] Example 1: Construction of a high-throughput bioanalytical system by reference to the first -4 Figure 'The method of manufacturing a bioanalysis system 1 will be described in detail below. First, a substrate 10 is formed and formed by a commercially available 0.25 μm semiconductor fabrication process for general logic and optical components. a plurality of photodetectors 21 on the upper surface of the substrate 1. The photodetector 210 is a photodiode photon detector each having an exposed area of 24 μm and a thickness of 452 μm. The photodetectors are arranged adjacent to each other such that an array of 512 rows and 512 columns of the photodetector 210 is formed on the substrate 10. "The plurality of control circuits 215 are formed on the upper surface of the substrate 10 without forming light. Detecting the position of state 210. In this embodiment, a control circuit 215 corresponds to an optical debt detector 210' to control the operation and control of the corresponding photodetector 210 between the optical detection device 210 and the recording device. 1378237 In this embodiment, a filter layer 240 is formed on the upper surface of the photodetector 210 and the control circuit 215. Before the filter layer 240 is formed, a global planarization process is performed on the upper surface of the photodetector 210 and the control circuit 215. Filter layer 240 includes a plurality of sublayers. In this embodiment, the germanium layer 240 can include a plurality of sublayers formed on a base substrate material that can be formed, placing, or deposited with a substrate material, such as SiO 2 on the substrate. 1〇 is available. In one embodiment, a semiconductor fabrication or deposition process can be implemented. For example, deposition methods may include, but are not limited to, physical deposition, chemical deposition, molecular beam epitaxy (MBE), and at mic layer deposition (ALD). In some embodiments, the substrate material can include one or more depressions formed on a surface. In some cases, the depressions may be non-planar, such as hemispherical or arcuate with a defined radius. For example, referring to Figure 3a, the radius of the sub-layer closest to the filter layer 240 of the substrate 10 in one embodiment may be about 6.1 μηη and may vary from 1 μπι to 100 μηη. The size of the recess in a system can vary depending on the design and application of the system and the size of the corresponding pinhole. In one embodiment, the radius of the system can be varied, for example, based on the application, design, characteristics, photodetector, and/or source of the system and the characteristics of the biomolecule to be detected. In some embodiments, the recess can be formed as part of the substrate formation process or by removing portions of the substrate material using conventional semiconductor process removal techniques. For example, the removal method may include wet etching, dry etching, or a method thereof. In other embodiments, a depression may be formed using pressure or pressure when the substrate material is formed. IS 3 Once the substrate material of filter layer 240 is formed as described above, a series of sublayers 40 1378237 ' can be formed on top of the substrate material to produce a filter layer ^ 240 as shown in Figure 3a. In some examples, a sub-layer can be formed by depositing a replacement layer of a material having different dielectric properties. For example, the filter layer 240 can be formed by first depositing a sub-layer having a higher refractive index on the surface of the planarized photodetector 21 and the control circuit 215 on the substrate material. Thereafter, a sublayer having a lower refractive index is deposited on the formed sublayer having a higher refractive index. In some embodiments, the sub-layer having a higher refractive index may comprise Nb 205, and the sub-layer having a lower refractive index may have Si 〇 2 . The tantalum layer 240 is formed by successively depositing a lower layer of higher refractive index and lower refractive index until a large number of sublayers have been deposited on the substrate material. In this embodiment, filter layer 240 includes 69 sublayers. The number of sublayers, their refractive indices, their materials, and/or other parameters can be varied based on various considerations, such as system applications, designs, features, photodetectors, and/or light sources. Referring to Figure 5, a table summarizing the construction of one of the filters, layer 240, is shown. In Fig. 5, a lower numbered sublayer provides a sublayer closer to the bottom surface of the filter layer 240, and a higher numbered sublayer provides a sublayer closer to the upper surface of the filter layer 240. As shown in Fig. 5, in this embodiment, the odd-numbered layers of the filter layer 240 are made of, for example, bismuth pentoxide (Nb205), which has a higher refractive index. The even sub-layer is made of, for example, SiO 2 (Si 〇 2), which has a lower refractive index. The sublayer can be formed by using a sputtering system, and examples of the sputtering system include Model No. RAS 110 of Radical Assisted Sputtering Series, by Shincron Co" 1^(1.(8110^8&\¥&-1 〇1, 丁〇1^〇, 八八)^). The thickness of each layer in this embodiment is also provided in the table of Figure 5. Synthetic filter layer 240 for the fluorescent group Cy5 It is highly transparent, while the light emitted from the [S] 41 1378237 (Helium-Neon) laser has a low penetration at a wavelength of about 633 nm, and the laser is used as an external light source to excite the fluorophore Cy5. Referring again to Figures 2 and 4, a masking sheet 230 having pinholes 235 is formed in the filter layer 240. In one embodiment, the pinholes 235 can be about 0.2 μm in size and can vary from 〇1 to 1 pm. The size of the system based design and application and the corresponding layer can vary depending on the pinhole size in the system. In one embodiment, based on many considerations, such as system applications, designs, features, photodetectors, and/or The light source and the characteristics of the biomolecule to be detected can change the radius. A masking sheet having a pinhole 235 is fabricated. A process of 230 on the filter layer 240 or the substrate 10 will be described in detail below. First, a photoresist layer is formed on the filter layer 240 by, for example, spin coating a photoresist layer (if filtering) The layer 24 is formed on the upper surface of the photodetector 210 and the control circuit 215 as needed or on the surface of the planarized photodetector 210 and the control circuit 215 (if the filter layer 240 is not formed). Thereafter, the photoresist is developed to form a photoresist pattern in the pinhole region. The pinhole region is covered by using a photomask, and the photoresist is exposed such that only the region covered by the photomask is maintained in the filter layer 240 or planarized. A photoresist pattern is formed on the upper surface of the photodetector 21 and the control circuit 215. The 'metal layer is then deposited on the filter layer 24 that has formed the photoresist pattern. In this embodiment, the metal layer may include chromium (Cr). By performing a magnetron sputtering process, it is deposited on the upper surface of the filter layer 24 or the planarized photodetector 210 and the control circuit 215. Then 'removed in the pinhole The portion of the metal layer on the region and the light 11 in the pinhole region and the pattern 'by forming a masking sheet 230 having a pinhole 235. Or 'by first depositing a metal layer (e.g., 'Cr) on the filter layer 240 ij 1 42 1378237 •, then forming a mask on the metal layer, thereby forming the metal layer A portion of the upper surface is exposed to form a masking sheet 230. The % portion of the exposed metal layer is then etched until the filter layer 240 is exposed, thereby forming pinholes on the metal layer. Thereafter, the mask is removed from the metal layer and a masking sheet 230 having pinholes 235 is formed on the filter layer 240. In some embodiments, the hemisphere formed by the space between the masking sheet 230 and the sub-layer closest to the masking sheet 230 is filled with SiO2, and the hemisphere is polished to a flat surface before the masking sheet 230 is formed. The metal layer has a pinhole 235 formed in the center of the half Φ ball of the filter layer 240. Referring again to Figures 2 and 4, in this embodiment, the joint location 220 is formed by filling a support material in the pinhole 235 or recess 450. The support material may be a polymer or inorganic material that is transparent to the fluorescent light emitted from the fluorescent group 36. Referring again to Figure 1, only 12 optical detection devices 20 are shown. It can be understood that at least 10,000 optical detecting devices 20 can be formed on the substrate 10. For example, in this embodiment, each of the optical detecting devices 20 has a circular shape having a radius of about 5 μm or less, which can occupy an area of about 100 μm 2 . For a substrate 10 having an area of 1 square inch (i.e., 2.54 cm by 2.54 cm), it is possible to construct more than six million optical detecting devices 20 on the substrate 10. By simultaneously operating those six million optical detection devices 20, biomolecules can be detected with high throughput. Example 2: Attachment and detection of biomolecules in high-throughput bioanalytical systems [S ] 43 1378237

一經螢光染劑Cy5標誌之核酸用來測試偵測系統。Cy5 與生物素分別附著至一 60員（mer)之寡核苦酸的3,與5, 端。將經標誌與生物素化之DNA溶解於TrisMg (1〇 mMOnce the fluorescent dye Cy5 is used, the nucleic acid is used to test the detection system. Cy5 and biotin are attached to the 3, and 5, respectively, of a 60-mer (mer) oligonucleotide. Dissociation of the labeled and biotinylated DNA into TrisMg (1〇 mM

Tris，10 mMNaCl，100 mM MgCl2, pH8)緩衝溶液中、將其 沈積於位置陣列上’並將其培養於一保濕箱（humid chamber) 中。約30分鐘後，以Tris緩衝溶液將未結合之DNA清洗 掉。 經由一 635 nm光散發二極體來提供激發光，635 nm 光散發二極體可形成於遮蔽薄板上。為了讀取來自各像素 之訊號’將激發光開啟約1 - 5秒’記錄來自各像素訊號， 且將此循環重複一百輪。之後相應地計算各像素之代表平 均訊號與對應之標準差。將DNA樣本沈積前與後之訊號進 行比較，且具有大於3倍標準差總和之平均訊號差異的像 素視為正(positive)像素，即， (Avg 後-Avg 前)> 3x(STD 後-STD 前)。 實施例3 :連結量子點至聚合酶 以下為將經官能化之量子點結合至一聚合酶分子上之 一級胺的兩種方法，第一種使用胺活化點（amine-activated dot)，第二種使用缓基活化點（carboxyl-activated dot)。 3.1胺EVITAGtm與DNA聚合酶之結合 經胺 EVITAG™ (例如 Evident Technologies, cat# E2-C11-AM2-0620 ; EVITAG™ 套組之 QD 產品也在[5 1378237 • eBioscience, Inc., San Diego，CA 之 eFluorTM 標誌下販售） • 官能化之量子點（QD)藉由BS3 (雙琥珀醯亞胺辛二酸酯納· 鹽（Bis(sulfosuccinimidyl) suberate))、一 同質雙功能 (homobifunctional)水可溶交聯劑來活化，其包含一胺反應 性N-羥基琥珀醯亞胺羧酸鹽（NHS)酯於一 8碳延長臂 (spacer arm)之各末端。在pH 7-9，NHS酯與QD表面上之 一級胺反應以形成穩定之醯胺鍵，且釋放NHS離去基團 (leaving group)。Taq DNA 聚合酶或 Phi29 DNA 聚合酶具 • 有許多一級胺（例如，離胺酸(K)殘基與各多胜肽之N端）， 其為可得之NHS酯交聯之標的。 3.1.1量子點之表面活化 以 25 μΐ 10 mM BS3(Bis(sulfosuccinimidyl) suberate), ?161^，？&11#2158〇)與25 011〇><?68(磷酸緩衝鹽，？117.4) 將2.0 nmol之EVITAG™活化於一具有dH20之終體積250 μΐ中。在培養30分鐘後，使用一 P10管柱(Amersham Biosciences，產品編號17-0851-01)將溶液去鹽且以lxPBS 洗提。有顏色之蛋白質包含經活化之QD。 3.1.2DNA聚合酶結合 藉由將於0.1 Μ碳酸鈉緩衝溶液中（pH 9.2)之100 之DNA聚合酶加入至混合物中來結合聚合酶。在混合均勻 後，培養於4°C並傾斜旋轉2小時。 3.1.3QD結合之聚合酶的純化 [S ] 45 1378237 藉由以 30K Microspin filter (Pall Corporation，part# OD100C33)在6000 i*pm離心5-10分鐘，將結合物濃縮至總 體積〜200 μΐ。藉由30K Microspin filter以dH20清洗結合 物兩次。 再來，以 Suprdex 30/75 Resin (GE Healthcare，part# 17-0902-10或17-1044-10用於小蛋白質與胜肽）藉由尺寸 排除來純化結合物。在將經濃縮之結合混合物載至管柱且 允許其進入柱床(column bed)前，以dH20預先平衡管柱。 在黑光激發下以dH20洗提這些樣本，並收集螢光片段。 將榮光片段加在一起且藉由以100K Microspin filter在 6000 rpm離心5-10分鐘濃縮至總體積〜1〇〇 μΐ。經純化與 濃縮之結合物可儲存於4°C。 3.2羧基DNA聚合酶之結合 經叛基 EVITAGtm(例如 Evident Technologies，cat# E2-C11-CB2-0620)官能化之QD經由ECD居中之硫-NHS 酯偶合反應來活化。胺反應性硫-NHS酯與例如在TaqDNA 聚合酶或Phi29 DNA聚合酶上之離胺酸(K)之侧鏈中之一 級胺反應。 3.2.1量子點之表面活化1 tsi 將 2.0 nmol 之 EVITAGtm 稀釋於 25 mM MES pH 5.0 緩衝溶液中。在立即使用前，EDC ( 1-乙基-(3-二曱基氨基 丙基）碳醯 二亞胺 鹽酸鹽 (l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide 46 1378237 hydrochloride))溶解於冷MES pH 5.0中以至濃度5〇 mg/m卜平行地’相似地製備於25 mMMES pH 5.0中之50 mg/ml 的硫-NHS(Pierce，part#24525)溶液。 之後’將50 μΐ之EDC溶液與50 μΐ之硫-NHS溶液加 至EVITAG™溶液。將混合物混合均勻並以慢傾斜旋轉於 室溫 30 分鐘。使用 一 ρι〇 管柱（Amersham Bi〇sciences，產 品編號17-0851-01)將溶液去鹽且以ο ι μ碳酸納緩衝溶液 (pH 9.2)來洗提。收集含經活化之qd的有顏色部分。 3.2.2DNA聚合酶結合 將於0.1 Μ碳酸鈉緩衝溶液中（pH 9.2)之100 的 DNA聚合酶加入至混合物中。在混合均勻後，將樣本培養 於4°C並傾斜旋轉2小時。 3.2.3與QD結合之聚合酶的純化 藉由以 30K Microspin filter (Pall Corporation，part# OD100C33)在6000 rpm離心5-10分鐘，將結合物濃縮至總 體積〜200 μ卜藉由30K Microspin filter以dH20清洗結合 物兩次。再來’以Suprdex 30/75 Resin藉由尺寸排除來純 化結合物。 簡單地說，以dH20預先平衡管柱《之後將經濃縮之 結合混合物載至管柱且允許其進入柱床(column bed)。在黑 光激發下以dH20洗提管柱，並收集螢光片段。將螢光片 段加在一起且藉由以100K Microspin filter在6000 rpm離 心5-10分鐘將其濃縮至總體積〜1〇〇 μ1。經純化與濃縮之 [S ] 47 1378237 結合物可儲存於4°C。 實施例4:連結聚合酶至元件 使用光一 NHS(photoNHS) ( N-經基琥拍酿亞胺竣酉楚 器連 分子（N-hydroxy succimido carboxylate)以一碳鏈連結 結至一疊氮墙基苯(azidonitrobenzene))以將一酵素，例如 聚合酶附著至一元件的兩種方法被描述。 4.1UV表面活化 藉由以UV光之光活化，一光NHS用來將一聚合酶附 著至於一裝置上之一經胺修飾的表面。UV光藉由排除氮激 起一疊氮硝基苯部分以產生高反應性氮烯(nitrene)基團。氮 烯與元件表面上之NH2反應以形成一聯氨（hydrazine)鍵。 連結器之另一端為NHS羧酸鹽，其與聚合酶上之離胺 酸殘基反應以產生一醯胺共價鍵。 4.1.1表面製備 將 1 mM 光 NHS (Sigma，Art No. A3407 ’ 分子里 390.35 )之溶液製備於95%乙醇中。清洗經胺修飾之表面 以碳酸鹽緩衝溶液且之後以95%乙醇。接著’將光NHS 溶液提供至經胺修飾之表面。254-365 nm UV光照射於表 面3分鐘，之後以95%乙醇沖洗三次。Tris, 10 mM NaCl, 100 mM MgCl2, pH 8) was deposited in a buffer solution on a positional array and cultured in a humid chamber. After about 30 minutes, the unbound DNA was washed away with a Tris buffer solution. Excitation light is provided via a 635 nm light emitting diode, and a 635 nm light emitting diode can be formed on the masking sheet. In order to read the signal from each pixel 'the excitation light is turned on for about 1-5 seconds', the signal from each pixel is recorded, and this cycle is repeated for one hundred rounds. Then, the representative average signal of each pixel and the corresponding standard deviation are calculated accordingly. The signal before and after the deposition of the DNA sample is compared, and the pixel having an average signal difference greater than 3 times the standard deviation is regarded as a positive pixel, that is, (Avg after -Avg before)> 3x (after STD - Before STD). Example 3: Bonding Quantum Dots to a Polymerase The following are two methods for binding a functionalized quantum dot to a primary amine on a polymerase molecule, the first using an amine-activated dot, the second A carboxyl-activated dot is used. 3.1 Amine EVITAGtm binding to DNA polymerase via amine EVITAGTM (eg Evident Technologies, cat# E2-C11-AM2-0620; EVITAGTM kit QD products are also available [5 1378237 • eBioscience, Inc., San Diego, CA • sold under the eFluorTM mark) • Functionalized quantum dots (QD) by BS3 (Bis (sulfosuccinimidyl suberate)), homogenous bifunctional (homobifunctional) water Activated by a dissolved crosslinker comprising an amine reactive N-hydroxysuccinimide carboxylate (NHS) ester at each end of an 8 carbon spacer arm. At pH 7-9, the NHS ester reacts with the primary amine on the surface of the QD to form a stable guanamine bond and releases the NHS leaving group. Taq DNA polymerase or Phi29 DNA polymerase has a number of primary amines (eg, an amino acid (K) residue and the N-terminus of each multi-peptide), which is the target for the cross-linking of available NHS esters. 3.1.1 Surface activation of quantum dots with 25 μΐ 10 mM BS3 (Bis(sulfosuccinimidyl) suberate), ?161^,? &11#2158〇) with 25 011〇><?68 (phosphate buffer salt, ?117.4) 2.0 nmol of EVITAGTM was activated in a final volume of 250 μM with dH20. After 30 minutes of incubation, the solution was desalted using a P10 column (Amersham Biosciences, product number 17-0851-01) and eluted with lxPBS. The colored protein contains activated QD. 3.1.2 DNA polymerase binding Polymerase was bound by adding 100% DNA polymerase in 0.1 Μ sodium carbonate buffer solution (pH 9.2) to the mixture. After mixing well, the culture was carried out at 4 ° C and tilted for 2 hours. 3.1.3 Purification of QD-bound polymerase [S] 45 1378237 The conjugate was concentrated to a total volume of ~200 μΐ by centrifugation at 6000 i*pm for 5-10 minutes with a 30K Microspin filter (Pall Corporation, part # OD100C33). The conjugate was washed twice with dH20 by a 30K Microspin filter. Further, the conjugate was purified by size exclusion using Suprdex 30/75 Resin (GE Healthcare, part # 17-0902-10 or 17-1044-10 for small proteins and peptides). The column was pre-equilibrated with dH20 before loading the concentrated binding mixture onto the column and allowing it to enter the column bed. These samples were eluted with dH20 under black light excitation and the fluorescent fragments were collected. The glory fragments were added together and concentrated by centrifugation at 6000 rpm for 5-10 minutes to a total volume of ~1 〇〇 μΐ with a 100K Microspin filter. The purified and concentrated combination can be stored at 4 °C. 3.2 Binding of Carboxy DNA Polymerase QD functionalized with a thiol EVITAGtm (e.g., Evident Technologies, cat# E2-C11-CB2-0620) is activated via an ECD-centered sulfur-NHS ester coupling reaction. The amine-reactive sulfur-NHS ester is reacted with, for example, a primary amine in the side chain of the lysine (K) on Taq DNA polymerase or Phi29 DNA polymerase. 3.2.1 Surface activation of quantum dots 1 tsi Equivalent 2.0 nmol of EVITAGtm was diluted in 25 mM MES pH 5.0 buffer solution. EDC (1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide 46 1378237 hydrochloride) was dissolved in cold before use immediately. A 50 mg/ml solution of sulfur-NHS (Pierce, part #24525) in 25 mM MES pH 5.0 was prepared similarly in MES pH 5.0 at a concentration of 5 〇 mg/m. Thereafter, 50 μL of EDC solution and 50 μL of sulfur-NHS solution were added to the EVITAGTM solution. The mixture was mixed well and spun at room temperature for 30 minutes at a slow tilt. The solution was desalted using a ρι〇 column (Amersham Bi〇sciences, product number 17-0851-01) and eluted with a 4.8 μM sodium carbonate buffer (pH 9.2). The colored portion containing the activated qd is collected. 3.2.2 DNA polymerase binding DNA polymerase 100 of 0.1 Μ sodium carbonate buffer solution (pH 9.2) was added to the mixture. After mixing well, the samples were incubated at 4 ° C and tilted for 2 hours. 3.2.3 Purification of polymerase bound to QD by centrifugation at 6000 rpm for 5-10 minutes with a 30K Microspin filter (Pall Corporation, part # OD100C33), the conjugate is concentrated to a total volume of ~200 μb by 30K Microspin filter The conjugate was washed twice with dH20. Again, the conjugate was purified by size exclusion with Suprdex 30/75 Resin. Briefly, the column is pre-equilibrated with dH20. The concentrated binding mixture is then loaded onto the column and allowed to enter the column bed. The column was eluted with dH20 under black light excitation, and the fluorescent fragments were collected. The fluorophores were added together and concentrated to a total volume of ~1 〇〇 μ1 by centrifugation at 6000 rpm for 5-10 minutes with a 100K Microspin filter. The purified and concentrated [S ] 47 1378237 conjugate can be stored at 4 °C. Example 4: Linking a polymerase to a component using a light-NHS (photoNHS) (N-hydroxy succimido carboxylate is bonded to a stack of nitrogen wall groups by a carbon chain) Azidonitrobenzene is described in two ways to attach an enzyme, such as a polymerase, to a component. 4.1 UV Surface Activation By activation with UV light, a photoNHS is used to attach a polymerase to an amine-modified surface on a device. The UV light excites the monoazide nitrobenzene moiety by excluding nitrogen to produce a highly reactive nitrene group. The nitrone reacts with NH2 on the surface of the element to form a hydrazine bond. The other end of the linker is an NHS carboxylate which reacts with an amino acid residue on the polymerase to produce a guanamine covalent bond. 4.1.1 Surface preparation A solution of 1 mM light NHS (Sigma, Art No. A3407 ' molecule 390.35) was prepared in 95% ethanol. The amine modified surface was washed with a carbonate buffer solution followed by 95% ethanol. The photo NHS solution is then provided to the amine modified surface. 254-365 nm UV light was applied to the surface for 3 minutes and then washed three times with 95% ethanol.

4·1·2 胺之末端蓋（en(j_cap) [S 48 1378237 將 l〇 mM 乙 k-N-破 j白酿亞胺酉旨（N_acet〇XySuccinimide) ； 之溶液製備於碳酸鹽緩衝溶液(pH 9.3)中且提供至表面以 末端蓋住任何未經反應之胺。於室溫以溫和搖晃培養此裝 置2小牯。接著清洗表面三次，每次以碳酸鹽緩衝溶液及 蒸館去離子水。 4.1.3DNA聚合酶結合 β再來，將於碳酸鹽緩衝溶液(ΡΗ9.3)中之1 mM聚合酶 #提供至表面且於持續溫和搖晃下在室溫培養2小時。接著 /月洗表面二次，每次以碳酸鹽緩衝溶液及pH 7 4 pBS (磷 酸緩衝鹽）。經聚合酶結合之表面可儲存在4。〇。 4.2經緩衝之表面活化 在另一實施例中，在緩衝環境下，可將光NHS活化與 結合至表面。之後在聚合酶存在下使用UV光以活化疊氮 φ 硝基笨部分。再次，高反應性氮烯在UV光下形成為一電 子不足基團（electron deficient group)且立即與聚合酶表面 - 上之一級胺反應，於表面上形成共價鍵結。 4.2.1表面製備 將 1 m]V[光 NHS (Sigma，Art No. A3407，分子量 390.35 )之溶液製備於碳酸鹽緩衝溶液中。經胺修掷之表 面以碳酸鹽緩衝溶液(pH 9.3)沖洗。將光NHS溶液提供至 經胺修飾之表面且於持續溫和搖晃下培養2小時。表面之 後以碳酸鹽緩衝溶液沖洗三次。 [S] 49 1378237 4.2.2胺之末端蓋 將製備於碳酸鹽緩衝溶液(pH 9.3)中之10 mM乙酸-N-號拍醯亞胺醋溶液提供至表面以末端蓋住任何未經反應之 胺基團。於室溫在持續溫和搖晃下培養此溶液2小時，且 之後以PBS緩衝溶液(pH 7.4)沖洗表面三次。 4.2.3DNA聚合酶結合4·1·2 amine end cap (en(j_cap) [S 48 1378237] The solution of l〇mM BK-N-acet〇XySuccinimide; prepared in carbonate buffer solution (pH 9.3) Provided to the surface to cover any unreacted amine at the end. Incubate the device for 2 hours at room temperature with gentle shaking. Then wash the surface three times, each time with carbonate buffer solution and steam deionized water. .3 DNA polymerase binds to β, and 1 mM polymerase # in carbonate buffer solution (ΡΗ9.3) is supplied to the surface and incubated for 2 hours at room temperature under gentle shaking. Then, the surface is washed twice a month. Each time with a carbonate buffer solution and pH 7 4 pBS (phosphate buffer salt). The polymer-bound surface can be stored at 4. 4.2 4.2 Buffered surface activation In another embodiment, in a buffered environment, The photoNHS can be activated and bound to the surface. UV light is then used in the presence of the polymerase to activate the azide φ nitro stupid moiety. Again, the highly reactive nitrene forms an electron deficient group under UV light (electron deficient) Group) and immediately with the polymerase surface - one level Amine reaction to form a covalent bond on the surface. 4.2.1 Surface preparation A solution of 1 m]V [photo NHS (Sigma, Art No. A3407, molecular weight 390.35) was prepared in a carbonate buffer solution. The surface was rinsed with a carbonate buffer solution (pH 9.3). The light NHS solution was supplied to the amine-modified surface and incubated for 2 hours with gentle shaking. The surface was washed three times with a carbonate buffer solution. [S] 49 1378237 4.2 .2 End cap of amine The 10 mM acetic acid-N-plyimide solution prepared in a carbonate buffer solution (pH 9.3) was supplied to the surface to cover any unreacted amine groups at the end. The solution was incubated for 2 hours with gentle shaking and then rinsed three times with PBS buffer (pH 7.4). 4.2.3 DNA polymerase binding

將1 mM DNA聚合酶之溶液製備於Pbs缓衝溶液(pH 7.4) 且提供至經末端蓋住之（611(|_(；叩06(1)表面。將1^光A solution of 1 mM DNA polymerase was prepared in a Pbs buffer solution (pH 7.4) and supplied to the end capped (611 (|_(;叩06(1) surface. 1^ light)

(254-365 nm)照射於表面3分鐘。之後以Pbs缓衝溶液(PH 7.4) 沖洗表面三次。經聚合酶結合之表面可之後儲存在 4°C。 實施例5:鹼基延伸定序形式 如上所討論’有兩種一般方法來辨認於逐步延伸時所 加之核苷酸：連續加入4種經相同性標誌之核苷酸或同時 加入4種經差別性標誌之核苷酸。各個這些形式的例子於 以下提供。 5.1連續加入4種具有相同性標諸之核脊酸(254-365 nm) was irradiated on the surface for 3 minutes. The surface was then rinsed three times with a Pbs buffer solution (pH 7.4). The surface bound by the polymerase can then be stored at 4 °C. Example 5: Base Extension Sequencing Forms As discussed above, there are two general methods for identifying nucleotides added during the stepwise extension: consecutive addition of four nucleotides of the same identity or simultaneous addition of four differences The nucleotide of the sex marker. Examples of each of these forms are provided below. 5.1 Continuous addition of 4 kinds of nuclear carboxylic acid with the same identity

5.1.1腺嘌呤(A)分子延伸：加入經阻礙與標諸之腺嘌 呤與合適之聚合酶於一定序反應緩衝溶液（例如，4〇 mM5.1.1 Adenine (A) Molecular Extension: Add a hindered and labeled adenine 呤 with a suitable polymerase in a sequence reaction buffer (eg, 4 mM

Tris-HCl pH 9.1, ImM MgCl2)中。只有當胸腺喷咬(τ)為在 50 1378237 被定序核酸中與末端連結引子之5’端鄰接之核苷酸時，將 腺嘌呤加至定序引子之3’端。若核酸之核苷酸最接近引子 之3’端為鳥嘌呤(G)、胞嘧啶(C)或腺嘌呤(A)，之後沒有延 伸會發生。 5.1.2延伸反應清潔步驟：在延伸反應完成之後，清洗 陣列晶片一次使用5xSSC與0.1% SDS，而一次使用 5xSSC以移除腺嘌呤與未反應之溶液。 5.1.3螢光偵測與記錄：於連結位讀取螢光訊號以確認 是否腺嘌呤已被延伸，其指出一對應之胸腺嘧啶於被定序 之核酸中。 5.1.4移除保護與螢光基團：於偵測後，藉由化學或酵 素裂解來移除保護與螢光基團。 5.1.5清潔步驟：清洗陣列晶片一次使用5xSSC與0.1 %SDS，而一次使用5xSSC以移除經裂解之保護與標誌基 團。 5.1.6校對與記錄：確認自先前延伸步驟成功移除保護 與螢光基團。若具有殘餘保護與螢光基團偵測，例如於偵 測與記錄系統2上之分析軟體會記錄位置。僅在下個清潔 步驟中確認保護與螢光基團移除時，定序反應之記錄才可 繼續。 51 1378237 5.1.7重複5.1.1-5.1.6，此次使用鳥嘌呤來用於延伸反 應。 5.1.8重複5.1.1-5.1.6，此次使用胞嘧啶來用於延伸反 應。 5.1.9重複5.1.1-5.1.6，此次使用胸腺嘧啶來用於延伸 反應。 5.1.10使用A、G、C、T之每四個延伸反應為一循環。 藉由重複循環，於一逐步方式中確認一核酸之序列。 5.2同時加入4種具有區別之標誌的核苷酸 5.2.1鹼基延伸反應：將4種經阻礙與有區別之標誌的 DNA核苷酸（A、G、C、T)與核酸聚合酶加入至一在陣 列上的定序緩衝溶液。延伸反應可只發生在定序引子之3’ 端。與最接近連結引子5’端之被定序核酸的核苷酸互補的 核苷酸，可被加至定序引子之3’端，參見，例如第6圖。 5.2.2延伸反應清潔步驟：在延伸反應完成之後，一次 以5xSSC與0.1% SDS，且一次以5xSSC來清洗晶片，以 移除在反應溶液中之殘餘材料。 [S ] 5.2.3螢光偵測與記錄：於各連結位偵測各有區別之螢 52 1378237 光訊號以確認所加入之核苷酸。 5.2.4移除保護與螢光基團：於偵測後，藉由化學或酵 素裂解來移除保護與螢光基團。 5.2.5清潔步驟：清洗晶片一次使用5xSSC與0.1% SDS，而一次使用VSSC以移除經裂解之保護與螢光基團。 5·2·ό校對與記錄：確認自先前延伸步驟成功移除保護 與螢光基團。若具有殘餘保護與螢光基團偵測，例如於偵 測與記錄系統2上之分析軟體會記錄位置。僅在下個清潔 步驟中確認保護與螢光基團移除時，定序反應之記錄才可 繼續。 5.2·7重複5.2.1-5.2.6。重複反應循環，以確認核酸之 序列。 實施例6 :已知核酸之定序 一化學合成之60員寡核苷酸，具有已知序列：生物素 -55-tcag tcag tcag tcag tcag tcag tcag tcag tcag tcag tcag tc ACACGGAGGTTCTA-3’，作為一定序模板。此定序模板與 一 14員之寡核苷酸定序引子（5’-TAGAACCTCCGTGT-3，） 結合。模板之5’端經修飾以包括一生物素分子。將模板附 著至一含有卵白素（streptavidin)之反應器表面。定序反應使 [s ] 53 1378237 用/ DNA聚合酶以執行一鹼基延伸反應於具有15 mM * DTT之1χ定序緩衝溶液中。各延伸反應步驟只有增加一形 - 式之核苷酸，其具有二級延伸保護（阻礙）基團與一螢光 標誌（例如’ Cy5)。若與定序引子之3,端鄰接之DNA模 板的核苷酸為與加入之核苷酸互補’則之後加入螢光標誌 鹼基。於清洗掉未反應之驗基材料後，偵測螢光訊號。若 所加入之驗基並非互補’則有螢光訊號會被彳貞測到。在偵 測後’化學移除螢光基團，且使用一含鹽溶液（例如 5xSSC ; 0.1% SDS)更進一步清洗陣列，且再偵測一次以 _ 確認螢光標誌之移除。在移除與清洗步驟之後，對於沒有 偵測到螢光訊號之位置而言，其被認為螢光標誌已移除。 於下個反應循環中獲得之營光訊號，之後被視為下個延伸 定序之訊號。在移除與清洗之後，對於仍然維持螢光訊號 之位置而言，其使用軟體被記錄為具有不完全移除反應。 來自位置之訊该:可持續以被記錄，只要確認於下個循環中 之移除步驟。基於此方法，可連續加入四種形式之反應鹼 基材料，並循裱地執行反應。因此，確認了模板之序列。籲| 雖然本發明已以較佳實施例揭露如上，然其並非用以 限定本發明’任何熟習此技藝者，在不脫離本發明之精 神和犯圍内’當可作些許之更動與潤飾，因此本發明之 保護範圍當視後附之申請專利範圍所界定者為準。 is 3 54 13/8237 【圖式簡單說明】 第1圖為一平面圖，其顯示一包括光學偵測裝置的一 陣列的生物分析系統。 ' 第2圖為沿著第1圖之線A_A之一剖面圖其顯示一 實施例之光學偵測裝置。 第3a圖為一剖面圖，其顯示根據一實施例之光學偵測 裝置的細部。 第3b圖為一剖面圖，其顯示根據另一實施例之光學偵 測裝置的細部。 第4圖為沿著第1圖之線A_ a的一剖面圖，其顯示另 一實施例之光學偵測裝置。 第5圖為一表格，其顯示一實施例之一濾層的構成。 第6圖顯示一連結於一元件之連結位的核酸。 第7圖顯示，在具有經阻礙與標誌之核苷酸的鹼基延 伸一循環後，一連結於一元件之連結位的核酸。 第8圖顯示於第7圖中所示之鹼基延伸定序反應的一 替代實施例。 第9圖顯示’藉由驗基延伸定序將許多核酸平行定序 的一循環。 【主要元件符號說明】 1〜生物分析系統； 2〜偵測與記錄系統，Tris-HCl pH 9.1, 1 mM MgCl2). Adenine is added to the 3' end of the sequencing primer only when the thymus squeezing (τ) is the nucleotide adjacent to the 5' end of the terminal linker in the 50 1378237 nucleic acid. If the nucleotide of the nucleic acid is closest to the 3' end of the primer, it is guanine (G), cytosine (C) or adenine (A), and no extension will occur thereafter. 5.1.2 Extension reaction cleaning step: After the extension reaction is completed, the array wafer is cleaned using 5xSSC and 0.1% SDS at a time, and 5xSSC is used at a time to remove adenine and unreacted solution. 5.1.3 Fluorescence Detection and Recording: Fluorescence signals are read at the junction to confirm whether adenine has been extended, indicating a corresponding thymine in the sequenced nucleic acid. 5.1.4 Removal of Protection and Fluorescent Groups: After detection, the protective and fluorescent groups are removed by chemical or enzymatic cleavage. 5.1.5 Cleaning Step: Clean the array wafer using 5xSSC and 0.1% SDS at a time, and use 5xSSC at a time to remove the cleavage protection and labeling groups. 5.1.6 Proofreading and Recording: Confirm that the protective and fluorescent groups were successfully removed from the previous extension step. If there is residual protection and fluorophore detection, for example, the analysis software on the detection and recording system 2 will record the location. The recording of the sequencing reaction can only continue if the protection and fluorophore removal are confirmed in the next cleaning step. 51 1378237 5.1.7 Repeat 5.1.1-5.1.6, this time use guanine for extension reaction. 5.1.8 Repeat 5.1.1-5.1.6, this time using cytosine for extension reactions. 5.1.9 Repeat 5.1.1-5.1.6, this time using thymine for the extension reaction. 5.1.10 Use four extension reactions of A, G, C, and T as one cycle. The sequence of a nucleic acid is confirmed in a stepwise manner by repeating the cycle. 5.2 Simultaneous addition of 4 different nucleotides with distinctive markers 5.2.1 Base extension reaction: Add 4 kinds of blocked and differentiated DNA nucleotides (A, G, C, T) and nucleic acid polymerase Up to a sequencing buffer solution on the array. The extension reaction can occur only at the 3' end of the sequencing primer. A nucleotide complementary to the nucleotide of the sequenced nucleic acid closest to the 5' end of the linker can be added to the 3' end of the sequencing primer, see, for example, Figure 6. 5.2.2 Extension reaction cleaning step: After completion of the extension reaction, the wafer was washed with 5xSSC and 0.1% SDS once, and once with 5xSSC to remove residual material in the reaction solution. [S ] 5.2.3 Fluorescence Detection and Recording: Detect each distinct firefly at each link 52 1378237 Optical signal to confirm the added nucleotide. 5.2.4 Removal of Protected and Fluorescent Groups: After detection, the protective and fluorescent groups are removed by chemical or enzymatic cleavage. 5.2.5 Cleaning Step: Clean the wafer using 5xSSC and 0.1% SDS at a time, and use VSSC at a time to remove the cleavage of the protective and fluorescent groups. 5·2·ό Proofreading and Recording: Confirmation that the protection and fluorescent groups were successfully removed from the previous extension step. If there is residual protection and fluorophore detection, for example, the analysis software on the detection and recording system 2 will record the location. The recording of the sequencing reaction can only continue if the protection and fluorophore removal are confirmed in the next cleaning step. 5.2·7 repeats 5.2.1-5.2.6. The reaction cycle is repeated to confirm the sequence of the nucleic acid. Example 6: Sequencing of a nucleic acid. A chemically synthesized 60-member oligonucleotide having a known sequence: biotin-55-tcag tcag tcag tcag tcag tcag tcag tcag tcag tcag tcag tc ACACGGAGGTTCTA-3', as a certain Sequence template. This sequencing template was combined with a 14-member oligonucleotide sequencing primer (5'-TAGAACCTCCGTGT-3,). The 5' end of the template is modified to include a biotin molecule. The template is attached to a reactor surface containing streptavidin. The sequencing reaction allowed [s] 53 1378237 to use /DNA polymerase to perform a one-base extension reaction in a 1 χ sequencing buffer solution with 15 mM * DTT. Each extension reaction step only adds a nucleotide of the formula, which has a secondary extension protection (obstruction) group and a fluorescent marker (e.g., 'Cy5). If the nucleotide of the DNA template adjacent to the 3rd end of the sequencing primer is complementary to the added nucleotide, then the fluorescent marker base is added. The fluorescent signal is detected after the unreacted test substrate material is washed away. If the test base is not complementary, then a fluorescent signal will be detected. After detection, the fluorophore is chemically removed and the array is further cleaned using a saline solution (e.g., 5xSSC; 0.1% SDS) and detected again to confirm the removal of the fluorescent marker. After the removal and cleaning steps, it is considered that the fluorescent marker has been removed for the location where the fluorescent signal is not detected. The camping signal obtained in the next reaction cycle is then considered as the next extended sequence signal. After removal and cleaning, the software used is recorded as having an incomplete removal reaction for the position where the fluorescent signal is still maintained. From the location message: Sustainable to be recorded, as long as the removal step in the next cycle is confirmed. Based on this method, four types of reaction alkali-based materials can be continuously added, and the reaction is carried out cyclically. Therefore, the sequence of the template is confirmed. The present invention has been disclosed in the above preferred embodiments, and it is not intended to limit the invention to any skilled person, and may make some modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of the invention is defined by the scope of the appended claims. Is 3 54 13/8237 [Simple Description of the Drawings] Fig. 1 is a plan view showing a bioanalysis system including an array of optical detecting devices. Fig. 2 is a cross-sectional view taken along line A_A of Fig. 1 showing an optical detecting device of an embodiment. Figure 3a is a cross-sectional view showing details of an optical detection device in accordance with an embodiment. Figure 3b is a cross-sectional view showing details of an optical detecting device in accordance with another embodiment. Fig. 4 is a cross-sectional view along line A_a of Fig. 1 showing an optical detecting device of another embodiment. Figure 5 is a table showing the construction of a filter layer of one embodiment. Figure 6 shows a nucleic acid linked to the junction of an element. Fig. 7 shows a nucleic acid linked to a junction of a member after a cycle in which the base of the nucleotide which is hindered from the marker is stretched. Figure 8 shows an alternative embodiment of the base extension sequence reaction shown in Figure 7. Figure 9 shows a cycle in which a number of nucleic acids are sequenced in parallel by base extension sequencing. [Main component symbol description] 1~Bioanalysis system; 2~Detection and recording system,

55 1378237 1 〇〜生物分析基板； 20〜光學偵測裝置； 210〜光偵測器； 220〜連結位； 215〜控制電路； 230〜遮蔽薄板； 235〜針孔； Η1〜距離； Η2〜距離； 240〜濾、層； 250〜微透鏡； 30〜生物分子； 32〜單股DNA分子； 344〜經修飾之核苷酸； 346〜黏合定序引子； 34〜末端引子； 36〜螢光團； 3 62〜核苦酸； 364〜螢光阻礙基團； 3 6Α〜第一位置； 36Β〜第二位置； 38〜聚合酶； 384〜方法； Θ1〜第一立體角； Θ2〜第二立體角； 56 1378237 Θ2〜第二立體角； 40〜激發光源； 410〜ρ型半導體層； 420〜光散發層； 430〜η型半導體層； 450〜凹處； 415、435〜金屬接觸； 440〜電源。55 1378237 1 〇 ~ biological analysis substrate; 20 ~ optical detection device; 210 ~ light detector; 220 ~ link position; 215 ~ control circuit; 230 ~ masking sheet; 235 ~ pinhole; Η 1 ~ distance; Η 2 ~ distance ; 240 ~ filter, layer; 250 ~ microlens; 30 ~ biomolecule; 32 ~ single strand of DNA molecule; 344 ~ modified nucleotide; 346 ~ adhesive sequence primer; 34 ~ end primer; 36 ~ fluorophore ; 3 62 ~ nucleotide acid; 364 ~ fluorescent blocking group; 3 6 Α ~ first position; 36 Β ~ second position; 38 ~ polymerase; 384 ~ method; Θ 1 ~ first solid angle; Θ 2 ~ second three Angle; 56 1378237 Θ 2 ~ second solid angle; 40 ~ excitation light source; 410 ~ p type semiconductor layer; 420 ~ light emitting layer; 430 ~ n type semiconductor layer; 450 ~ recess; 415, 435 ~ metal contact; power supply.

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Claims (1)

1378237 第 98135549 號七、申請專利範圍： 修正日期:101.8.171378237 No. 98135549 VII. Patent application scope: Date of amendment: 101.8.17 f多正本 1. 一種鑑定單一生物分子之裝置，包括： 一基板，具有一光偵測器，該基板被設置來偵測散發 自該生物分子的光； 一遮蔽薄板(blind sheet)於該基板上，該遮蔽薄板包括 一針孔，該針孔具有一直徑； 一濾層，其被提供於該遮蔽薄板與該基板之間且被提 供於該針孔之下’該濾層為非平面且被設置來過濾散發自 該生物分子的光；以及 一連結位(linker site) ’其被提供接近於該針孔，該連 結位被處理以將該生物分子置於接近該針孔。 2_如申請專利範圍第1項所述之鑑定單一生物分子之 裝置，其中該光偵測器係擇自電荷耦合元件 (charged-coupled device)、互補金屬氧化半導體 (complementary metal-oxide semiconductor, CMOS)感測器 或光二極體(photodiode)其中之一。 3.如申請專利範圍第1項所述之鑑定單一生物分子之 裝置’其中該非平面包括弧線形、球形、橢圓形或弓形其 中之一。 4 ·如申睛專利範圍弟1項所述之鑑定单·一生物分子之 裝置’其中該針孔具有一小於或等於約1，〇〇〇 nm之直徑。 5·如申清專利耗圍弟1項所述之鑑定单·一生物分子之 裝置，其中該針孔具有一小於或等於約200 nm之直徑。 6. —種鑑定單一生物分子之裝置，包括： 58 1378237 第98135549號. 修正日期:101.8.17 修正本 * 一基板，具有一光偵測器，該基板被設置來偵測散發 . 自該生物分子的光； 一光散發層形成於該基板上，其中該光散發層包括一 凹處； 一濾層，其被提供於該凹處與該基板之間且於一針孔 之下，該濾層為非平面且被設置來過濾散發該生物分子的 光；以及 一連結位，其被提供接近於該凹處與該針孔，該連結 • 位被處理以將該生物分子置於接近該針孔。 7. 如申請專利範圍第6項所述之鑑定單一生物分子之 裝置，其中該光散發層穿過該凹處沿著平行於該光偵測器 之一表面的水平方向散發來自一激發光源的光至該連結 位。 8. 如申請專利範圍第6項所述之鑑定單一生物分子之 裝置，更包括一遮蔽薄板於該基板上，該遮蔽薄板包括該 針孔，該針孔具有一直徑。 * 9.如申請專利範圍第6項所述之鑑定單一生物分子之 -裝置，其中該非平面包括弧線形、球形、橢圓形或弓形其 中之一。 10. 如申請專利範圍第6項所述之鑑定單一生物分子之 裝置，其中該針孔具有一小於或等於約l，〇〇〇nm之直徑。 11. 如申請專利範圍第6項所述之鑑定單一生物分子之 裝置，其中該針孔具有一小於或等於約200 nm之直徑。 12. 如申請專利範圍第6項所述之鑑定單一生物分子之 裝置，其中該激發光源係擇自一發光二極體（light emitting 59 1378237 第98135549號 修正日期:101.8.17 修正本 diode，LED)、一有機發光二極體（〇rganic Hght emitting diode, OLED)、一尚分子發光二極體（polymer light emitting diode，PLED)與一雷射二極體 Qaser di〇de，LD)。 13. —種光學偵測系統’包括至少1〇,〇〇〇個如申請專利 範圍第1項所述之裝置。 14. 一種光學偵測系統’包括至少25〇,〇〇〇個如申請專 利範圍第1項所述之裝置。 15 · —種光學偵測系統’包括至少2,〇〇〇,〇〇〇個如申請 專利範圍第1項所述之裝置。 16_—種光學偵測系統’包括至少1〇,〇〇〇,〇〇〇個如申請 專利範圍第1項所述之裝置。 17. —種鑑定生物分子之襞置，包括： 一基板’具有一光偵測器； 一遮蔽薄板於該基板上，該遮蔽薄板包括一針孔之陣 列； 一濾層，形成於該針孔之陣列之下，其中被提供於各 個之至少一些該針孔之下的一濾層結構為非平面；以及 一連結位形成接近於各針孔，該連結位被處理以使該 生物分子附於該連結位。 18. 如申請專利範圍第17項所述之鑑定生物分子之裝 置，其中該光偵測器係擇自電荷耦合元件、互補金屬氧化 半導體感測器或光二極體其中之一。 19. 如申請專利範圍第17項所述之鑑定生物分子之裝 置，其中該非平面包括弧線形、球形、橢圓形或弓形其中 1378237 • 第98135549號 修正曰期:ΗΠ.8.Ι7 修正本 . 20.如申請專利範圍第17項所述之鑑定生物分子之裝 ' 置，其中各針孔具有一小於或等於約1,000 nm之直徑。 « ' 21.如申請專利範圍第17項所述之鑑定生物分子之裝 置，其中各針孔具有一小於或等於約200 nm之直徑。 22. —種光學偵測系統，包括至少1〇,〇〇〇個如申請專利 範圍第6項所述之裝置。 23. —種光學偵測系統，包括至少250,000個如申請專 ' 利範圍第6項所述之裝置。 Ιφ 24.—種光學偵測系統，包括至少2,000,000個如申請 專利範圍第6項所述之裝置。 25. —種光學偵測系統，包括至少10,〇〇〇,000個如申請 專利範圍第6項所述之裝置。 26. 如申請專利範圍第1項所述之鑑定單一生物分子之 裝置，其中該光以一正交角度穿過該濾層。 27. 如申請專利範圍第6項所述之鑑定單一生物分子之 裝置，其中該光以一正交角度穿過該濾層。 • 28.—種鑑定一單一分子的方法，包括： ， 將一生物分子附著至一連結位，該連結位被提供於接 近形成在一遮蔽薄板上的一針孔，該針孔具有一尺寸； 就由一滤層來過濾一散發自該生物分子的光，該濾、層； (i)被提供於該遮蔽薄板與一基板之間，該基板具有一 光偵測器， .... (ii)被提供於該針孔之下，以及 (iii)為非平面；以及 使用該光偵測器來偵測穿過該濾層的光。 61A multi-original device 1. A device for identifying a single biomolecule, comprising: a substrate having a photodetector, the substrate being configured to detect light emitted from the biomolecule; and a blind sheet on the substrate The masking sheet includes a pinhole having a diameter; a filter layer is provided between the masking sheet and the substrate and is provided under the pinhole. The filter layer is non-planar and Light is provided to filter light emitted from the biomolecule; and a linker site 'which is provided proximate to the pinhole, the joint is processed to place the biomolecule close to the pinhole. 2_ The device for identifying a single biomolecule as described in claim 1, wherein the photodetector is selected from a charge-coupled device, a complementary metal-oxide semiconductor (CMOS) One of a sensor or a photodiode. 3. The device for identifying a single biomolecule as recited in claim 1, wherein the non-planar surface comprises one of an arc, a sphere, an ellipse or an arc. 4. The device of claim 1, wherein the pinhole has a diameter less than or equal to about 1, 〇〇〇 nm. 5. The device of claim 1, wherein the pinhole has a diameter less than or equal to about 200 nm. 6. A device for identifying a single biomolecule, comprising: 58 1378237 No. 98135549. Amendment date: 101.8.17 Revision * A substrate having a photodetector, the substrate being arranged to detect emissions. a light of a molecule; a light emitting layer is formed on the substrate, wherein the light emitting layer comprises a recess; a filter layer is provided between the recess and the substrate and under a pinhole, the filter The layer is non-planar and is configured to filter light that emits the biomolecule; and a link that is provided proximate to the recess and the pinhole, the link being processed to place the biomolecule close to the needle hole. 7. The device for identifying a single biomolecule according to claim 6, wherein the light emitting layer passes through the recess and emits light from an excitation source in a horizontal direction parallel to a surface of the photodetector. Light to the link. 8. The device for identifying a single biomolecule of claim 6, further comprising a masking sheet on the substrate, the masking sheet comprising the pinhole, the pinhole having a diameter. * 9. The device for identifying a single biomolecule as described in claim 6 wherein the non-planar comprises one of an arc, a sphere, an ellipse or an arc. 10. The device for identifying a single biomolecule of claim 6, wherein the pinhole has a diameter less than or equal to about 1, 〇〇〇nm. 11. The device for identifying a single biomolecule of claim 6, wherein the pinhole has a diameter of less than or equal to about 200 nm. 12. The device for identifying a single biomolecule according to claim 6, wherein the excitation light source is selected from a light emitting diode (light emitting 59 1378237, No. 98135549, date of revision: 101.8.17, revision of the diode, LED ), an organic light emitting diode (OLED), a polymer light emitting diode (PLED) and a laser diode Qaser di〇de, LD). 13. An optical detection system' comprising at least one device, such as the device of claim 1 of the scope of the patent application. 14. An optical detection system' comprising at least 25 turns, such as the device of claim 1 of the patent application. 15 - An optical detection system 'includes at least 2, 〇〇〇, a device as described in claim 1 of the patent application. The 16_-type optical detection system 'includes at least 1 〇, 〇〇〇, 装置 a device as described in claim 1 of the patent application. 17. An apparatus for identifying biomolecules, comprising: a substrate having a photodetector; a masking sheet on the substrate, the masking sheet comprising an array of pinholes; a filter layer formed in the pinhole Under the array, a filter layer provided under at least some of the pinholes is non-planar; and a joint is formed adjacent to each pinhole, the joint being processed to attach the biomolecule The link. 18. The device for identifying a biomolecule according to claim 17, wherein the photodetector is selected from one of a charge coupled device, a complementary metal oxide semiconductor sensor, or a photodiode. 19. The device for identifying biomolecules according to claim 17, wherein the non-planar comprises an arc, a sphere, an ellipse or an arch, wherein 1378237 • No. 98135549 is amended: ΗΠ.8.Ι7 Amendment. 20 The device for identifying biomolecules according to claim 17, wherein each pinhole has a diameter of less than or equal to about 1,000 nm. The apparatus for identifying biomolecules of claim 17, wherein each pinhole has a diameter of less than or equal to about 200 nm. 22. An optical detection system comprising at least one device, such as the device of claim 6 of the scope of the patent application. 23. An optical detection system comprising at least 250,000 devices as described in claim 6 of the application. Ι φ 24. An optical detection system comprising at least 2,000,000 devices as described in claim 6 of the scope of the patent application. 25. An optical detection system comprising at least 10, 〇〇〇, 000 devices as described in claim 6 of the scope of the patent application. 26. The device for identifying a single biomolecule of claim 1, wherein the light passes through the filter layer at an orthogonal angle. 27. The device for identifying a single biomolecule of claim 6, wherein the light passes through the filter layer at an orthogonal angle. • 28. A method of identifying a single molecule, comprising: attaching a biomolecule to a bonding site, the bonding site being provided adjacent to a pinhole formed in a masking sheet, the pinhole having a size; A filter layer is used to filter a light emitted from the biomolecule, the filter layer; (i) is provided between the shielding sheet and a substrate, the substrate has a photodetector, .... Ii) being provided under the pinhole, and (iii) being non-planar; and using the photodetector to detect light passing through the filter layer. 61